Often, one of the first signs of trouble with a liquid chromatography (LC) method is a failure to pass the system suitability requirements for the method. In fact, early warning of potential problems is one of the primary purposes of a system suitability test. As stated in the regulatory guidance (1), "The accuracy and precision of HPLC data collected begin with a well-behaved chromatographic system. The system suitability specifications and tests are parameters that provide assistance in achieving this purpose." Unfortunately, sometimes we lose sight of the purpose of a system suitability test and worry more about checking the boxes on a form than what the test is trying to do. I often am asked what system suitability requirements are mandated by regulation. This is an interesting question, because as far as I know, there are no firm requirements as to what parameters must be measured or what the minimum values of these parameters must be. The closest thing that I can find to requirements is a document called "Reviewer Guidance: Validation of Chromatographic Methods," (1) from the Food and Drug Administration's Center for Drug Evaluation and Research (FDA-CDER). This is a document intended to give CDER reviewers of LC methods some guidelines about what to look for in a "good" method. Or as the document states (1), "The purpose of this technical review guide is to present the issues to consider when evaluating chromatographic test methods from a regulatory perspective." Many workers feel that if their methods perform at least as well as the recommendations of this document, they will be safe from regulatory criticism. This may or may not be a well-reasoned response. This month's "LC Troubleshooting" discussion will center on the recommendations of the CDER document, especially in terms of what it means from a practical method performance standpoint. 

Table I summarizes four of the key parameters that can be used to evaluate method performance. These are standard measurements that most workers make on a routine basis. Let's review the definitions of each of these, then look at some examples. 

Table I: Minimum system suitability recommendations1

. The retention factor (also sometimes called capacity factor, 

') is a measure of the distribution of the analyte between the mobile phase and the stationary phase in the column in isocratic (constant organic solvent) separations. It can be thought of as a way to measure retention in a manner that is independent of column dimensions and flow rate. Retention factor is calculated as 

 is the retention time of the analyte of interest and 

 is the column dead-time ("solvent front"). As has been discussed in other "LC Troubleshooting" columns (for example, see reference 2), when developing a new method, we strive for 2 < 

 < 20 for all the peaks of interest in a separation. Peaks with 

 < 1 can be compromised with real samples that contain large "garbage" peaks at 

 > 2 also tend to be more robust to small changes in the chromatographic conditions. 

. Resolution is the measurement of the separation of two peaks in a chromatogram and is a function of both the peak widths 

where the subscripts refer to the first and second peaks. The peak width is determined by drawing tangents to the sides of the peak and measuring the distance between the tangents where they intersect the baseline. Baseline separation of a perfectly shaped Gaussian peak pair (a rarity in LC) is observed with 

 = 1.5 (see peaks 1 and 2 in Figure 1a). 

. Peak tailing is measured with the tailing factor in the pharmaceutical industry, and with a slightly different calculation called the asymmetry factor 

 for most nonpharmaceutical applications.  

 is the width of the peak at 5% of its height and 

 is the front half-width at the same height. (

 is calculated as the back half-width divided by the front half-width at 10% of the peak height.) New columns with well-behaved test compounds will generate tailing factors of 0.9–1.2, so a little tailing is normal. 

. The plate number, also called the column efficiency, is a unitless way to measure the performance of the column. It is calculated as: 

Plate numbers for new columns can be quite large. For example, a 150 mm × 4.6 mm column packed with 5-μm diameter particles can give plate numbers of 12,000–15,000 with well-behaved compounds. A 100 mm × 4.6 mm, 3-μm column might give 13,000–16,000 plates under the same conditions. Under more realistic conditions with "real" samples, plate numbers of 10,000 are more reasonable for these columns, and we will use this as a reference value for a well-behaved 150 mm × 4.6 mm, 5-μm or 100 mm × 4.6 mm, 3-μm column in the present discussion. 

Let's look next at the practical impact of the recommendations of Table I. Figures 1 and 2 contain simulated chromatograms that will help illustrate various points. A 150 mm × 4.6 mm, 5-μm particle diameter column operated at a flow rate of 1 mL/min is assumed (

 = 10,000; in Figure 2, the plate number is noted. Tailing is simulated using an exponential calculation, which might be adequate for some types of tailing, but not others. 

 = 1.75 for peak 4. It can be seen that with these chromatograms with a fairly small 

 > 1 should avoid any interference with the unretained material. However, if the unretained peak is quite large and tails badly, it is quite possible that retention of 

 < 1 might have some interference problems. So the recommendation of Table I of 

 > 2 is fairly conservative; although it is nice to have 

 > 1, the first peak should be sufficiently retained to avoid problems with the unretained peak. Sometimes 

-values are reduced to speed up the separation, but this can be false economy, because of potential for problems at the beginning of the chromatogram. One way around this problem is to increase the flow rate, which will shorten the retention times but will not affect 

 — most methods operate well below the maximum pressure limits of the LC system, so an increase in flow (and pressure) can be tolerated. 

In Figure 1a, symmetric peaks are assumed (

 = 1.0) and the peak spacing has been adjusted so that the resolution between peak 1 and peak 2 is 1.5, between peak 2 and peak 3 is 1.7, and between peaks 3 and 4 is 2.0. For these perfectly shaped peaks, it can be seen that with 

 = 1.5, the valley between the peaks just touches the baseline. If there is any deterioration of the column or peak tailing, the valley will rise and resolution will be inadequate. Many workers accept 

 = 1.7 as a minimum (the separation of peaks 2 and 3), and although this does offer baseline separation, there is no safety margin if the column deteriorates. Only 

 = 2.0 (between peaks 3 and 4) gives both adequate separation and a little safety for small changes in the separation.  

When the peaks tail, resolution suffers. This is illustrated in Figure 1b for 

 = 2.0. With no change in retention times, now the resolution that ranged from 1.5 to 2.0 in Figure 1a is only 1.0 to 1.3 in Figure 1b, and 0.75 to 1.0 in Figure 1c. This means that if peaks tail and 

 > 2.0 is desired, retention must be increased, which will result in longer run times (see discussion of Figure 2). Tailing peaks also complicate the calculation of resolution. With equation 2, symmetrical peaks are assumed. If peaks tail, it is only the portion of the peak that contributes to the valley that is important, so the back half of the first peak and the front half of the second peak contribute to the resolution. Data systems base resolution calculations on the peak width, so reported values with tailing peaks might not reflect the true resolution. 

Peak width changes with the square root of the plate number (equation 4), so smaller plate numbers mean broader peaks and less resolution. Figure 2 shows the result of a combination of 

 = 1.5 for various plate-number columns. As 

 is reduced, retention must be increased to maintain resolution. To obtain 

 = 1.5 for all peaks, the run time of Figure 1b must be increased to that shown in Figure 2a. The run must be even longer for columns that have lower plate numbers and, thus, generate broader peaks, as seen by comparing the chromatograms of Figure 2. The larger retention times and lower plate numbers combine to give shorter peaks (constant area is assumed), so detection limits will suffer when the plate number is reduced. For example, The retention of peak 4 is 50% larger in Figure 2c when compared with Figure 2a. At the same time, the peak width is more than twice as large, so the peak height and, thus, sensitivity has dropped by more than twofold. 

So are the recommendations of Table I reasonable or not? I have mixed feelings about using these values as targets for system performance, but they certainly are not unreasonable. If you can develop a method so that 

 > 2 for the first peak, you are likely to have fewer problems with the method, but many methods work very well with 

 > 1. The remaining parameters are somewhat interrelated. As columns age, typically peak tailing increases and the plate number drops. These factors, coupled with any change in retention time, will result in a change in resolution — usually for the worse. For this reason, it usually is worthwhile to spend extra time during method development to develop a more robust separation — one that will tolerate the inevitable changes in the separation that occur over time.  

 = 2 is adequate for equal-sized peaks. If the peak size varies widely between peaks in the chromatogram, such as with impurity analysis or stability-indicating assays, larger values of resolution might be required. Today's high-purity-silica columns generate much more symmetrical peaks than the previous generation of columns, so I would target a maximum tailing factor of 1.5–1.7. More tailing than this usually can be avoided by picking another column, adjusting the mobile phase pH, or changing the mobile phase organic solvent. Remember, if you develop a method that starts with 

 = 2, it will only get worse as the column ages.  

A plate number of 2000 is likely to be inadequate unless you have a very simple separation. You will be able to get better sensitivity, higher resolution, and faster runs (all other factors the same) with a column that generates more plates. A new 150-mm-long, 5-μm particle or 100 mm, 3-μm column will give 

  10,000 for real compounds; by the time the initial plate number drops by 25–30%, it is probably time to replace the column. Although a new column costs $500 or so, if it lasts more than 500 injections, it accounts for less than 2% of the overall analysis costs, so it is prudent to replace the column when it gives signs of deterioration. 

If you meet the guidelines of Table I, will your methods always pass regulatory scrutiny? No. Is it necessary to meet the guidelines to have a method that performs well? No. However, if you use Table I as it is intended — a guideline — you should be able to develop methods that perform well and will pass regulatory inspection. The key is to test the method for robustness — tolerance to small changes of the type expected in normal operation, such as ±2% organic, ±3 °C, or ±0.1 pH units. And then develop a system suitability test that really checks to be sure the method is functioning adequately to gather reliable data. If you develop a robust method and document how and why you chose the method settings, you will be less likely to have regulatory issues. More importantly, you will understand the effects of the method variables on method performance, so you can keep the method working well and can know what to do when it begins to show signs of problems. 

"LC Troubleshooting" Editor John W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of LCGC's editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of LC trouble-shooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at 

(1)  "Reviewer Guidance: Validation of Chromatographic Methods," FDA-CDER (1994), 

Articles

LC Troubleshooting

This month's "LC Troubleshooting" discussion will center on the recommendations of the CDER document, especially in terms of what it means from a practical method performance standpoint.

Testing Method Performance

When one is approached to write an article about the history of high performance liquid chromatography (HPLC), it is both a compliment and a vivid reminder that you have been around a long time. One of the first things that I recalled was a project that started about 10 years ago with Dave Carr and Mike Henry, who have been well known HPLC contributors over the years. In 1997, Dave and Mike decided to preserve some history by interviewing as many chromatographers as possible who were around during the early days of HPLC. Their plan was to publish a book about the interesting history of HPLC before too much first-hand information was lost. The book was never published, but I felt that some of the material gathered by Dave and Mike might be of interest to the many readers of such a widely circulated magazine as 

. Dave Carr dug into his archives and was able to locate a first-person account supplied by Dr. Jack Kirkland (1) about the early days in HPLC from his perspective. Those days corresponded to my own, so I was pleased to find a record for comparison. To keep my dates consistent, I also referred to an article about Jack authored by Klaus Unger (2) as I wrote this historical account from my own memory and perspective. 

When modern HPLC was emerging, I was just beginning my own professional career as a new DuPont employee at the Experimental Station in Wilmington, Delaware, where Jack worked. I graduated from Penn State University (University Park, Pennsylvania) in 1966, where I did thermal analysis under Professor Joseph Jordan and shared laboratories with Peter Carr, who also later converted to HPLC and made important contributions to its development. I conducted my first HPLC experiments as a postdoctoral student in 1966–1967 at Purdue University (West Lafayette, Indiana) with Professor Lockhart Burgess (Buck) Rogers, who was always one of the first people to recognize the importance of a new analytical technique. Interestingly, I shared labs at Purdue with Ron Majors and Brian Bidlingmeyer, who were graduate students in liquid separations. The fact that I have remained involved with HPLC from its beginning is due in large part to the many cordial relationships that I have enjoyed over the years. My HPLC career can be divided into three parts: the early years at Dupont, the middle years in California, when I traveled extensively and met many scientists in Europe and Japan, and the later years when I returned to Pennsylvania to teach at Penn State and start Keystone Scientific. This article will focus on the early years. Perhaps I will have a chance to write more about the others in the future. 

I arrived at the DuPont Experimental Station in 1967 as a research chemist at Carothers Lab. It was named after Wallace Carothers, who invented the first true synthetic fiber, nylon, in 1931. I immediately joined the Analytical Discussion Group that met regularly in the Experimental Station cafeteria to share exciting analytical results. One of the first lunch talks was given by Jack Kirkland, who was already a respected analytical chemist within DuPont, as well as an expert in the still relatively new technique of gas chromatography (GC). Because he worked in the Biochemicals Department, there were many problems that could not be solved by GC because of the thermal instability of the solutes. In 1964, Jack visited the laboratories of Josef Huber in Europe, where important HPLC research was being conducted, and he recognized the value of this technique for his work back at DuPont. Jack became convinced that better particles would unlock the LC performance predicted in the early 1960s by Cal Giddings, John Knox, Josef Huber, and other scientists, so he returned to the Experimental Station and immediately gained approval to conduct research aimed at creating improved particles for HPLC. His stimulating talk that day was about a new superficially porous particle; it was named Zipax, which rolls off the tongue much more easily than superficially porous. Zipax was invented in collaboration with Ralph Iler, a DuPont scientist who many considered to be the father of silica and silicone chemistry (3). Zipax spheres had a particle size of 30–40 μm and showed higher efficiency than other available HPLC particles due to their uniform size and a thin porous diffusion layer, but they were hampered by low surface area that could support only about 1% stationary phase (w/w). After hearing Jack's lunch talk, I became very excited by the promising new HPLC technology and wanted to become part of it, even though I knew that a satisfying career like Jack's in analytical separation would be very hard to achieve at Carothers Lab. I was further inspired at that time by a visit to Paul Hamilton at nearby A. I. DuPont Institute (Wilmington, Delaware), where Paul was achieving very impressive amino acid separations on small particle ion-exchange resins (4). By 1969, I had transferred from Carothers Lab to become one of the early employees of the newly formed DuPont Instrument Products Division (IPD) located just a few miles south of the Experimental Station in Glasgow, Delaware. Before leaving DuPont in 1973, I continued to work closely with Jack at the Experimental Station along with other scientists at IPD, including Joe DeStefano, John Larmann, Bob Moody, and others. This group of friends later formed Rockland Technologies and continued to build upon the success of another product developed by Jack, the small, totally porous Zorbax HPLC particles. 

When I arrived at IPD, I vividly remember finding a nearly empty HPLC laboratory except for three things: a prototype HPLC instrument called the Model 820 (Figure 1), a large box full of customer samples, and a bottle of Zipax particles to coat with liquid phase. At that time, we used primarily β, β'-oxydipropionitrile (abbreviated ODPN or BOP for obvious reasons) as the liquid stationary phase; the ODPN-coated Zipax was tap-packed into straight 50 cm × 2.1 mm columns. Injection was made with GC syringes though a septum that lasted for only a few hours once pierced, even though septa were made with the best DuPont fluorocarbon elastomer technology. The Model 820 HPLC instrument was quite innovative at the time because it featured a 3000 psi pneumatic pump that had a higher pressure rating than most other HPLC instruments available in the early 1970s. Gas amplifier pumps operate at constant pressure and were an excellent choice because they did not require leak-free seals and check valves for steady flow and good quantitation. Our choice of a pneumatic pump was even more important because a leaking septum during the run did not destroy the integrity of the analytical data for that sample; flow would hold very constant, even with various small system leaks, because column inlet pressure was controlled. The biggest disadvantage of constant-pressure HPLC instruments was that flow diminished and peak areas increased when the column became even slightly plugged; retention time had to be monitored carefully to know when this was happening. As in GC, use of internal standards was important for accurate quantitation. Pneumatic pumps are still important in HPLC, although more convenient, electromechanical metering pumps have replaced them in most, but not all, HPLC instruments. Hardware milestones achieved at DuPont IPD in the early 1970s included the development of a unique, low-dwell-volume gradient device that blended digitally and accurately at high pressure after the pump. Another significant hardware development at IPD was the replacement of the septum injector with a loop injection valve. In the early 1970s, Stan Stearns of Valco (then headquartered in Houston, Texas) visited our IPD laboratory, where we successfully tested a GC valve that had been modified to operate at higher pressure for HPLC; Stan and I have continued to maintain a productive relationship over the years. 

Unfortunately, columns with a liquid phase coating usually lasted only about a day or two before losing too much retention as the immiscible (not insoluble) liquid phase gradually moved from column to waste reservoir. Gradients were impossible with these early "stationary" phases, so polymer coatings were developed that improved column stability and even allowed gradient elution. Three important polymer phases, Zipax SCX, Zipax SAX, and Zipax HCP, emerged from DuPont's exceptional polymer technology and became very popular in the 1970s. The first success, Zipax SCX, was the subject of Kirkland U.S. Patent 3,577,266, issued on May 4, 1971. It used a coating of Nafion or sulfonated tetrafluorethylene copolymer discovered in the late 1960s by Walther Grot of DuPont; it had important HPLC applications for herbicides and other cationic solutes. Zipax SCX would have no doubt become popular in the pharmaceutical industry, but HPLC had not yet been accepted by the FDA. Zipax SAX was made from a quaternized hydrophobic polymer that was an ingredient in hair spray designed to preserve the rigid hair styles of the times; Zipax SAX was excellent for separation of anionic solutes and became one of the first HPLC packings accepted by the FDA for routine analysis of food dyes. Zipax HCP, an abbreviation for hydrocarbon polymer, was one of the very first commercial phases to employ reversed-phase conditions. It was made from a rubber-like, ethylene–propylene copolymer designed for garden hoses and other premium applications in which stability and flexibility over a range of temperatures was needed; Zipax HCP was an excellent general purpose phase for all hydrophobic solutes and quickly became the preferred column in my toolbox, much like C18 phases are today. During this same time period, Jack also developed and patented one of the first highly stable bonded phases based upon silicone chemistry. Called Zipax Permaphase, it made our life in the laboratory much easier, especially when coupled with gradient elution and an injection valve. Permaphase ODS (C18) and ETH (Ether) demonstrated that stable covalent bonding would be the direction for all future HPLC particles; however, we continued to solve numerous problems with polymer-coated Zipax while we developed improved silica particles and additional bonded phases. 

Although Zipax and other superficially porous particles (some were then called pellicular), such as Corasil I and II from Waters (Milford, Massachusetts) and Perisorb from E. Merck (Darmstadt, Germany), were important stages in the evolution of HPLC in the late 1960s and early 1970s, the surface areas were too low (1–10 m

/g); solute retention was barely adequate for many solutes. Small porous spheres were being developed that had much higher surface areas. Just before I left DuPont in 1973, I had the chance to work with Zorbax, another one of Jack's creations that used Ralph Iler's silica–polymer aggregation (coacervation) technology during synthesis. The first Zorbax particles were 7 μm in size and had 60-Å pores with a surface area of 300 m

/g. Although Zorbax was the first commercially available porous spherical particle to my knowledge, porous spherical particles such as Spherisorb, Hypersil, and others also were being developed in Europe. Long columns filled with superficially porous particles were supplanted quickly by shorter, more retentive columns filled with high-efficiency, porous silica. 

The history of HPLC is primarily about the history and evolution of particle technology. While instrumental developments such as improved pumps, gradient formers, valves, and detectors have all been important, most of the real innovations that have propelled the HPLC technique to prominence have been related to HPLC particles and columns. Figure 2 depicts the history of HPLC particle technology, in which important new developments have occurred on a regular basis for more than 40 years. While porous layer particles made a brief appearance in the 1960s and early 1970s, the steady trend has been to reduce particle size to improve efficiency, as shown in Figure 3. Better column efficiency slows the rate of band spreading and creates narrower peaks, allowing columns to deliver higher resolution and peak capacity. More efficiency also enables more speed and less solvent consumption because shorter columns can give the same separation as longer, larger particle columns. Efficiency increases inversely with particle diameter, as shown in Figure 4, and resolution only increases with the square root of efficiency. Because pressure drop increases inversely with the square of particle diameter, the cost of increasing resolution using smaller particles is very high. Every time particle size is reduced significantly, another round of instrument development usually must occur to handle the pressure. In 1965, Cal Giddings wrote about the effect of mean particle diameter on HPLC column performance (5), "The foregoing arguments indicate that a zero particle diameter is the optimum. However, very little resolution gain is found beyond a certain reduction in 

, rapidly becomes neglible. Thus, a point is reached in reducing 

 where there is very little theoretical advantage, and severe practical disadvantage to further reduction. The practical disadvantages stem from the excessive pressure drop needed to force mobile fluid through the column and the difficulty of preparing a uniform packing of extremely fine materials." Cal was already explaining that the C-term alone would not be able to deliver better small molecule performance below a certain particle size and was pointing to the A-term or flow uniformity for additional gains. 

Jack Kirkland's latest HPLC particles (6–8) might be his most impressive accomplishment because he has been able to achieve almost twice the efficiency of 3-μm particles without a significant increase in operating pressure. Figure 5 shows an electron micrograph of cross-sectioned 1.7-μm solid-silica spheres that have been coated with a 0.5-μm porous layer. The 2.7-μm porous-layer particle retains about 75% of the surface area and sample capacity of a porous particle. The most remarkable attribute of this particle is that it shows comparable efficiency to a much smaller porous particle in the 1.7–1.8 μm range, as shown in Figure 6. While higher efficiency with porous-layer particles has been clearly demonstrated for larger molecules that benefit greatly from reducing the path of diffusion into the particle (that is, reducing the C-term of the van Deemter equation), the extremely high efficiency of the new particles is surprising for small molecules. The theoretical explanation for the remarkable performance of these denser particles can lie partly in their very narrow particle size distribution, which in turn greatly improves flow uniformity along the column (reducing the A-term of the van Deemter). Called Fused-Core, the new porous-layer particles are produced by Advanced Materials Technology (Wilmington, Delaware) and are offered commericially by Mac-Mod Analytical (Chadds Ford, Pennsylvania) under the tradename Halo and by Supelco Division of Sigma-Aldrich (Bellefonte, Pennsylvania) under the tradename Express. I am happy to be associated with Jack again in my capacity as a consultant for Supelco. 

 Edition 1 (John Wiley & Sons, Incorporated, New York, 1979). 

Dynamics of Chromatography, Part I. Principles and Theory

 (Marcel Dekker, Inc., New York, 1965), p. 281. 

(6) J.J. DeStefano, T.J. Langlois, and J.J. Kirkland, 

(8) J. DeStefano, private communication. 

I would like to thank Ron Majors for asking me to write this article and also thank Supelco Division of Sigma-Aldrich and Advanced Materials Technology for supplying certain Figures. 

The History of Chromatography

Guest columnist Dick Henry traces the history of HPLC back more than 40 years, starting with his days at DuPont.

The Early Days of HPLC at DuPont

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This issue completes the multi-part MS primer with a list of terms derived from common usage throughout various industries and, while included with the MS Primer. is intended to stand alone. A number of current books recognize the need to monitor and modify the language we use within LC–MS. When a practice such as LC–MS becomes so diverse and prolific the terms and phrases adopted from various sources finding a way into the practice need scrutiny. Many books such as the "Mass Spectrometry Desk reference" (2nd edition, O. David Sparkman, paperback 198 pages published by Global View Publishing, June 30, 2006) provide more than a description of fundamentals making an attempt to explain and unify usage. 

Although I prefer IMMS there may be little problem in most cases determining that 'IMS' means ion mobility mass spectrometry and not imaging mass spectrometry let alone standing as a reference to Dr. Jed Diamond's irritable male syndrome (although I'm sure cases could be made). 

My thanks to everyone – I look forward to the comments posted in the margin at 

 (Resource Library/Primers). The web version and the hardcopy (due out in early 2009) have clearly benefit from your contributions. 

The following list of terms is derived from common usage throughout the industry as an adjunct to the discussions in this primer and includes terms and techniques no longer in common usage. 

 When viewed as similar to absorbance displayed on a UV detector, the vertical increase in signal above background indicates an increased occurrence of that particular ion (when the 

 axis is calibrated in mass units) or total ions present (when the horizontal axis is calibrated in time or scans). The signal for all ions resulting from the fragmentation of a single analyte or compounds compared to a base peak (the relative abundance of each ion) is used to determine the fit of a fragmented pattern to a library spectrum for positive identification. 

 The measured mass value for a compound with an associated error like 5 ppm. Accurate mass also is used commonly to refer to the technique rather than the measured mass. Exact mass is the exact theoretical value for the mass of a compound.  

Atmospheric solids analysis probe (ASAP):

 Based upon work by Horning in the 1970s, this form of sample ionization developed by McEwen and McKay uses a standard atmospheric pressure chemical ionization (APCI) plasma but forms ions by placing the sample in a heated nitrogen stream. The heat volatilizes a surprisingly large number of samples, and ions are formed by charge exchange with metastable ions created by the APCI plasma. Relatively unambiguous identifications can be made of individual compounds from complex mixtures at low levels using accurate mass instruments. See also DART and DESI. 

 The term used to refer generally to techniques such as electrospray ionization (ESI) and APCI and others that operate at atmospheric pressure. 

Atmospheric pressure chemical ionization (APCI):

 Originally called solvent-mediated electrospray, it is applied successfully more often to neutral molecules that do not ionize easily directly out of solution. APCI provides a current on a sharp pin, positioned in the on-coming aerosol stream, to create a plasma of metastable ions from the solvent itself and transfer the charge from these ions to the analyte as it passes through the plasma. Heating a probe through which the liquid chromatography (LC) or solvent stream passes creates the aerosol. 

Developed by Charles McEwen at DuPont in 2002. Using a heated transfer line, a standard gas chromatography (GC) effluent can be introduced to a standard API (or ESI–APCI) source on a mass spectrometer. This provides an easy and fast changeover from ESI to gas chromatography (GC) for compounds that would be best analyzed by GC. Mode of ionization can be either APCI or atmospheric pressure photoionization (APPI). 

Atmospheric pressure photoionization (APPI):

 Developed in the 1980s but commercialized after 2000 when krypton gas lamps were found to generate sufficient photon energy at 10 eV (approximately) to ionize nonpolar analytes such as PAHs and steroids not typically amenable to ESI and APCI ionization. 

 Usually the most intense peak in the spectrum to which others are compared; in ionization techniques that give extensive structural information such as electron ionization (EI), the base peak may not be the parent or molecular ion. 

 Substances of known mass are introduced usually as a constant flowing stream while the mass spectrometer software acquires a signal for a given set of filtering conditions (that is, RF/DC ratio for a quadrupole instrument). After comparing the acquired signal to a reference file, a calibration look-up table is created in the software. The calibration table is then the basis for the mass-to-charge ratios passed by the quads to be assigned a specific value. See the MS Primer sections on "Quantitation and Calibration." 

 Related to electrospray ionization; a theory first proposed in 1968 by Malcolm Dole in which he hypothesized that as a droplet evaporates, its charge remains unchanged. The droplet's surface tension, ultimately unable to oppose the repulsive forces from the imposed charge, explodes into many smaller droplets. These Coulombic fissions occur until droplets containing a single analyte ion remain. As the solvent evaporates from the last droplet in the reduction series, a gas-phase ion forms. 

Collisions induced at low vacuum (0.4 torr) by the introduction of a reagent for the purpose of enhancing the production of molecular ions and often sensitivity; as this is a much lower energy process than electron impact ionization, fragmentation is reduced and it is often referred to as a soft ionization technique. See electron ionization. 

Also referred to as collisionally activated dissociation (CAD), it is a mechanism by which molecular ions are fragmented in the gas phase by acceleration (using electrical potential) to a high kinetic energy in the vacuum region followed by collision with neutral gas molecules such as helium, nitrogen or argon. A portion of the kinetic energy is converted or internalized by the collision, which results in chemical bonds breaking and the molecular ion is reduced to smaller fragments. Some similar "special purpose" fragmentation methods include electron transfer dissociation (ETD) and electron-capture dissociation (ECD). See the MS Primer section on "Biomolecular ionization methods." 

 Developed by Robert Cody and others in 2002, similar to desorption electrospray ionization (DESI) in application although more closely related to APCI in function. A sample is placed on a substrate and bombarded by energized particles formed in a process similar to APCI. That is, metastable ions are formed by a plasma and transported by heated nitrogen gas directed at the target. See also McEwen's work listed under atmospheric GC and ASAP. 

 Developed for matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) instruments, it "cools" and focuses the ions for approximately 150 ns after they form and before accelerating the ions into the flight tube. The cooled ions have a lower kinetic-energy distribution than uncooled ones, and they ultimately reduce the temporal spread of the ions as they enter the TOF analyzer, resulting in increased resolution and accuracy. DE is significantly less advantageous with macromolecules (for instance proteins >30,000 Da). 

Desorption electrospray ionization (DESI):

 First described by Graham Cooks in 2002 as a means of producing soft secondary ions from (typically) an inert substrate surface. Analogous to MALDI using an ESI probe aimed at about 50° incident angle to the surface allowing ions to chemically sputter and be admitted to the mass spectrometer. Shown to produce information directly from many polar and nonpolar surface materials (skin, intact fruit for pesticide residue detection, and so forth) without the need for sample preparation. See also McEwen's work listed under atmospheric GC and ASAP. 

 Once viewed as an alternative to a preparing samples in a MALDI substrate, especially for small molecules because the substrate (a bare silica surface) would not generate interfering ions. Its commercial potential in the late 1990s diminished as the difficulties of producing the plates and the surface susceptibility to contamination became apparent. 

 For our interests, the term usually is used in conjunction with "radio frequency" (RF) when describing how the quadrupole functions as a mass filter. Superimposed RF and constant DC potentials between four parallel rods were shown by Wolfgang Paul in 1953 to act as a mass separator, or filter, where only ions within a particular mass range, exhibiting oscillations of constant amplitude, could collect at the analyzer.  

 Sometimes incorrectly referred to as "electron impact" ionization resulting from the interaction of an electron with a particle (atom or molecule); can be thought of as a "hard" ionization technique, as sufficient energy is imparted to disrupt internal chemical bonds requiring high kcal/mol. Ionizing voltage (typically 70 eV) refers to the difference in voltage causing acceleration of the electrons used to induce electron ionization. Unlike CI, EI avoids uncontrolled collisions by operating at high vacuum. The analyzer operates at even higher vacuum (10

 A so-called "soft" ionization technique. The most widely employed of the API techniques. Commercially significant since the late 1980s, the phenomenon is attributed to an excess of energy (voltages in the 3–5 kV range) applied to a conductive tube (stainless steel capillary) inducing the liquid flowing inside to exceed its Rayleigh limits and forming an aerosol upon exiting the tube. The resulting spray (the result of a coulombic explosion) then gives rise to ions contained in the aerosol droplets as they desolvate to approximately a 10-μm radius. The ions typically are protonated and detected in the form M+H in positive ionization mode or M-H in negative ion mode. 

 The nominal mass of an ion, molecule, or radical is the sum of the nominal masses of the elements in its elemental composition. Achieving accurate mass measurement is based upon the calculated elemental composition but the term "elemental analysis" is performed typically on inorganic materials — to determine elemental makeup, not structure — in some cases using solid metal samples. Inductively coupled plasma (ICP) sources are common where a discharge (or lower power-glow discharge) device ionizes the sample. Detection using dedicated instruments, at the parts-per-trillion level, is not uncommon. 

 The exact theoretical value for the mass of a compound. "Accurate mass" is the measured mass value for a compound with an associated error like 5 ppm. Accurate mass also is used commonly to refer to the technique rather than the measured mass. 

 One of the earlier so-called soft ionization techniques where the result is usually an intense molecular ion with little fragmentation. The analyte is placed in a matrix (often glycerol) either flowing or more commonly placed on the tip of a probe and positioned in the path of high-energy atoms — often xenon or cesium iodide. The technique has been effective for biomolecules up to 10,000 amu, but perhaps more importantly, in conjunction with the magnetic sector mass spectrometer, where exact weight can be determined such as for novel peptides. Sensitivity can be very good (low femtomole levels). The technique can be difficult to master, as the glycerol does foul the mass spectrometer source and the lower masses can be obscured by the presence of glycerol ions. This technique is little used now since the introduction of ESI. 

 The soft ionization provided by FI leads to little or no fragmentation for a wide variety of analytes. This is particularly significant in petrochemical applications, in which there are limitations to the utility of other ionization techniques due to fragmentation (EI) or complex ionization characteristics (CI). A thin wire with a high applied voltage is heated in the vapor of an organic compound such as indene. The resulting structures that are deposited on the surface of the wire, dendrites, are pyrolized, producing very fine conductive filaments. When a very fine point has a high potential applied to it, a very intense electric field is generated at this tip, providing conditions under which field ionization can take place. 

Sample molecules pass in close proximity to the tips of a mass of carbon dendrites grown on the FI emitter. The FI emitter is positioned in close proximity to a pair of hollow extraction rods. The emitter is held at ground potential and a relatively high voltage (12 kV) is applied to the rods, producing very high electric fields around the tips of the carbon dendrites. The GC column is positioned in close proximity and in line with the emitter wire. Under the influence of the electric fields, quantum tunneling of a valence electron from the molecule takes place to give an ion radical. 

 This is the practice of introducing a sample (usually purified in an earlier step, as a fraction to remove interferences and complexity from the resulting spectrum) through the LC injector, but without a column in-line. The LC system acts as a sample introduction device only. 

 In EI the filament is the source of electrons that interact with the analyte to ionize it. Typically made from a metal wire (flat or round) capable of giving off 70-eV electrons as it heats from current passing through it. 

 An ion produced as the loss from a parent molecular ion. The sum of the dissociated fragments equals the parent and under given conditions will always fragment the same internal bonds to produce a predictable pattern (same ions and relative abundances for each). See also product (daughter) ions resulting from specific MRM experiments. 

 Analytes must be converted from their resting state to an ion in order to be manipulated and acquired by a mass spectrometer. There are a number of ways to accomplish this as described in the primer — some more aggressively creating fragments while others conserve the analyte intact. Energy is presented to the analyte, producing an ion in the gas phase as opposed to, for instance, the condensed phase used to separate analytes in LC. 

 Usually refers to an instrument that is a combination of two different types, for instance, earlier micromass "hybrids" combined magnetic sector and quadrupoles. Today's quadrupole time-of-flight (QTOF) instrument is a hybrid of quadrupole and TOF. 

 Mass spectrometers can only manipulate and therefore detect a mass when it possesses at least one charge. When only one charge is present (for instance, by the loss of an electron, causing the molecule to exist as a positively charged cation radical or by the addition of a proton or hydrogen to exist as a positively charged pseudomolecular ion), we can think of it as representing the molecular weight in a low-resolution scheme. 

 The electric current detected based upon the charged particles created in the ion source. If the mass spectrometer is set to scan over a range of 100–500 Da, the resulting total ion current will be the sum of all ions present in the source within that range at the selected time. If the instrument is set to detect only one ion (selected ion monitoring), the resulting total ion current will be the sum of only that ion at each selected instance. 

 Cyclotron instruments trap ions electrostatically in a cell using a constant magnetic field. Pulses of RF voltage create orbital ionic motion, and the orbiting ions generate a small signal at the detection plates of the cell (the ion's orbital frequency). The frequency is related inversely to the ions' mass-to-charge ratio (

), and the signal intensity is proportional to the number of ions of the same 

 in the cell. At very low cell pressures, a cyclotron instrument can maintain an ion's orbit for extended periods, providing very high resolution measurements. Fast-Fourier transform-ion cyclotron resonance (FT-ICR) instruments represent the extreme capability of measuring mass with the ability to resolve closely related masses. Although impractical for most applications, a 14.5-T magnet can achieve a resolution of more than 3.5 million and, thus, display the difference between molecular entities whose masses vary by less than the mass of a single electron. 

Reference to ESI; in 1976, Iribarne and Thomson proposed the ion evaporation mechanism (see also "charge residue mechanism"), in which small droplets form by Coulombic fission, similar to the way they form in Dole's charge residue model. However, according to ion evaporation theory, the electric field strength at the surface of the droplet is high enough to make leaving the droplet surface and transferring directly into the gas phase energetically favorable for solvated ions.  

 This technique differentiates ions based upon a combination of factors: their size, shape, and charge, as well as their mass. Ion mobility measurements and separations coupled with tandem mass spectrometry (MS) can help overcome analytical challenges that could not be addressed by other analytical means, including conventional MS or LC instrumentation. Ion mobility mass spectrometry (or IMMS because "imaging mass spectrometry" is often abbreviated IMS) devices are used commonly in airports and handheld field units for rapidly (20 ms) detecting small molecules whose mobility is known. A wide range of analytes are amenable to IMMS. 

 The physical space in the ion stream in front of the analyzer, where the analyte is ionized. Each type of interface requires its own internal geometry for optimum results. 

 Also linear trap and Q-trap (or quadrupole trap). Wolfgang Paul's invention of the quadrupole and quadrupole ion trap earned him the Nobel Prize in physics in 1953. An ion trap instrument operates on principles similar to those of a quadrupole instrument. Unlike the quadrupole instrument, however, which filters streaming ions, the trap stores ions in a three-dimensional space. Before saturation occurs from too many ions attempting to fill the finite space, the trap or cyclotron allows selected ions to be ejected for detection. Fields generated by RF voltages applied to a stacked or "sandwich" geometry (end-cap electrodes at opposing ends) trap ions in space between the two electrodes. Ramping or scanning the RF voltage ejects ions from their secular frequency, or trapped condition. Dynamic range is sometimes limited. The finite volume and capacity for ions limits the instrument's range, especially for samples in complex matrices. 

The ability to perform sequential fragmentation and, thus, derive more structural information from a single analyte (that is, fragmenting an ion, selecting a particular fragment, and repeating the process) is called MS

 . GC peaks are not wide enough to allow more than a single fragmentation (MS-MS or MS

 ). Ion-trap instruments perform MS-MS or fragmentation experiments in time rather than in space, like quadrupole and sector instruments. So they cannot be used in certain MS-MS experiments such as neutral loss and precursor ion comparisons. Also, in MS-MS operation with an ion-trap instrument, the bottom third of the MS-MS spectrum is lost, a consequence of trap design. To counter the loss, some manufacturers make available via their software wider scan requirements that necessitate the switching of operating parameters during data acquisition.  

Because of similarities in functional design, quadrupole instruments are hybridized to incorporate the advantages of streaming quadrupole and ion trapping behavior to improve sensitivity and allow on-the-fly experiments not possible with either alone. Such instruments are sometimes called linear traps or Q-traps). The increased volume of a linear trap instrument (over a three-dimensional ion trap) improves dynamic range. 

 Although often presumed to be constant and stable, natural isotope abundance ratios show significant and characteristic variations when measured very precisely. Isotope ratio measurements are useful in a wide range of applications, for example, metabolic studies using isotopically enriched elements as tracers; climate studies using measurements of temperature-dependent oxygen and carbon isotope ratios in foraminifers; rock age dating using radiogenic isotopes of elements such as lead, neodymium or strontium; and source determinations using carbon isotope ratios (for instance to determine if a substance occurs naturally or is a petroleum-based synthetic). 

Typically, single focusing magnetic sector mass spectrometers with fixed multiple detectors (one per isotope) are used. Complex compounds are reduced to simple molecules before measurement — for example, organic compounds are combusted to CO

 Compounds can be dissolved in a material that acts as an intermediate to transfer charge to the analytes of interest. A laser is aimed at the mixture to cause sputtering to produce ions in the space just above the mixture, where they can be sampled or drawn into the mass spectrometer. Especially useful as a very soft ionization technique to look at intact large molecules because the low-mass ions from the matrix produce a highly complex, intense background that could otherwise interfere with analytes of similar low mass.  

 Ions leaving the ion source are accelerated to a high velocity; they then pass through a magnetic field perpendicular to their direction. When acceleration is applied perpendicular to the direction of motion, the object's velocity remains constant, but the object travels in a circular path. Therefore, the magnetic sector is designed as an arc. Ions with a constant kinetic energy but different mass-to-charge ratio are brought into focus at the detector slit (called the "collector slit") at different magnetic field strengths. 

The magnetic sector alone will separate ions according to their mass-to-charge ratio. However, because the ions leaving the ion source do not have exactly the same energy (and therefore do not have exactly the same velocity), the resolution will be limited. An electric sector that focuses ions according to their kinetic energy usually was added, which, like the magnetic sector, applies a perpendicular force to the direction of ion motion.  

Matrix assisted laser desorption ionization (MALDI):

 First introduced in 1988 by Tanaka, Karas, and Hillenkamp, MALDI uses a laser to strike and energize a matrix containing the analyte. It has proven to be the method of choice for ionizing exceptionally large peptide and protein molecules that can then be detected intact. Commonly employed as the introduction scheme for TOF instruments, which are often referred to as MALDI-TOF. 

MS– The Practical Art

This column completes the multipart MS primer with a list of terms derived from common usage throughout various industries.

A Mass Spectrometry Primer, Part IV

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a unique group of neurodegenerative diseases of the central nervous system, which include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease (CWD) in deer, and Creutzfeldt–Jacob disease (CJD) in humans. TSEs are caused by the conversion of a normal cellular prion protein designated PrP

  (1,2) and are characterized by spongiform degeneration, reactive astrocytosis and prion protein accumulation in the central nervous system. Although the species barrier provides significant protection from the interspecies transmission of prion disease, the BSE epidemic and the resulting rise in variant CJD (vCJD) illustrates the potential impact of prion disease upon human and economic health. Current validated diagnostics for prion diseases are all postmortem. The "gold standard" in prion diagnosis is immunohistochemistry utilizing antiprion protein antibodies on the obex region of the brain (3). Despite the good specificity and sensitivity of these tests, animals infected with prion disease only can be diagnosed late in the preclinical period when sufficient abnormal PrP

In recent years, there has been a growing interest in applying mass spectrometry (MS)-based proteomics technology to research on biomarker discovery and clinical diagnostics of diseases such as cancers and neurodegenerative disorders. Body fluids such as plasma and cerebrospinal fluid are rich sources of biomolecules. Investigation of these proteomes can reveal correlations between altered protein expression and certain disorders. The glycoproteome is one of the major subproteomes of blood, where glycoproteins secreted into the bloodstream comprise a major part of the plasma proteome. Functionally, the oligosaccharide moieties of various glycoproteins act as selectivity determinants, playing a fundamental role in many biological processes such as immune response and cellular regulation because cell-to-cell interactions involve sugar–sugar or sugar–protein specific recognition (4). Consequently, aberrant glycosylation has now been implicated in many diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular disease, and cancer (5).  

Here we present a method that enables isolation, identification, and relative quantification of glycoproteins in blood samples using lectin affinity enrichment, isotopic labeling, and tandem MS. By applying this method, 280 glycoproteins were identified from mouse plasma, among which 49 showed greater than twofold changes in the infected animals compared with the controls. Many of the identified proteins are known to be present at very low abundance, which demonstrates the ability of this method to delve into the plasma proteome.  

 C57/Bl6 mice were inoculated intraperitoneally with 50 μL of 10% brain homogenate from infected or control mice. At 108 days post-inoculation, animals were anesthetized with isoflorane and blood was collected by cardiac puncture into EDTA-treated vacutainer tubes. The whole blood was centrifuged at 1000xg for 5 min. Plasma was decanted and immediately frozen in liquid nitrogen. Samples from seven infected and seven control mice were pooled separately.  

 Tris hydrochloride, chicken ovalbumin, N-acetyl-D-glucosamine, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, manganese chloride tetrahydrate, formaldehyde, deuterated formaldehyde, and sodium cyanoborohydride were purchased from Sigma-Aldrich (St. Louis, Missouri). Sodium chloride, calcium chloride, sodium acetate, and urea were obtained from Fisher Scientific (Pittsburgh, Pennsylvania). Agarose-bound concanavalin A (Con A, 6 mg lectin/mL gel) and wheat germ agglutinin (WGA, 7 mg lectin/mL gel) were purchased from Vector Laboratories (Burlingame, California). Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega (Madison, Wisconsin). Iodoacetamide was obtained from MP Biomedicals (Solon, Ohio).  

 Lectin affinity columns were prepared by adding 400 μL each of Con A and WGA slurry to empty Micro Bio-Spin columns (Bio-Rad Laboratories, Hercules, California). The flow through the spin columns was driven by an Eppendorf 5415D centrifuge. A 40-μL volume of pooled mouse plasma was spiked with 4 μg of chicken ovalbumin and was diluted to 400 μL with the binding buffer (20 mM Tris, 0.15 M sodium chloride, 1mM Ca

 , pH 7.4) and was loaded on the lectin affinity column. After shaking for 6 h, the flow-through containing the unretained proteins was discarded and the lectin bead–protein complexes were washed with 2.5 mL of binding buffer. The captured glycoproteins were eluted with 2 mL of elution buffer (10 mM Tris, 0.075 M sodium chloride, 0.25 M N-acetyl-D-glucosamine, 0.17 M methyl-α-D-mannopyranoside, and 0.17 M methyl-α-D-glucopyranoside). The eluted fraction was concentrated by ultrafiltration using a 10-kDa Centricon Ultracel YM-10 filter (Millipore, Billerica, Massachusetts).  

 The concentrated sample was denatured with 6 M urea in 0.2 M sodium acetate buffer, pH 8, and reduced by incubating with 10 mM DTT at 37°C for 1 h. The reduced proteins were alkylated for 1 h in darkness with 40 mM iodoacetamide. The alkylation reaction was quenched by adding DTT to a final concentration of 50 mM. The samples were diluted to a final concentration of 1 M urea. Trypsin was added to the sample at a 50:1 protein to trypsin mass ratio. The sample was incubated at 37 °C overnight.  

 Sodium cyanoborohydride was added to the protein digest to the final concentration of 50 mM. The control sample was labeled with 0.2 mM formaldehyde, while the infected sample was labeled with 0.2 mM deuterated formaldehyde. The mixture was vortexed, incubated at 37 °C for 1 h. A 2 M ammonium hydroxide solution was added to quench the reaction and the mixture was dried immediately in Speedvac. The samples were reconstituted in 100 μL of doubly distilled water.  

High-pH reversed-phase liquid chromatography:

 Aliquots (50 μL each) of light-labeled control sample and heavy-labeled infected sample were combined and injected onto a Rainin (Varian, Palo Alto, California) high performance liquid chromatography (HPLC) system with a high pH–stable reversed-phase column (150 mm × 2.1 mm, 3 μm Gemini C18, Phenomenex, Torrance, California) at a flow rate of 150 μL/min. The peptides were eluted with a 40-min gradient consisting of 5–45% buffer B (buffer A: 100 mM ammonium formate, pH 10; buffer B: acetonitrile). Fractions were collected every 3 min for 60 min. Collected fractions were dried by Speedvac and reconstituted in 20 μL of 0.1% formic acid. LC–MS-MS analysis was performed using 2-μL injections of each of the 13 fractions containing peptides.  

 A nanoLC system (Eksigent, Dublin, California) was used to deliver the sample to a trap column and the solvent gradient (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile). Samples were flushed with mobile phase A at 5 μL/min for 3 min and loaded onto a self-packed column (100 mm × 0.15 mm, 3 μm ProSphere HP C18, Grace Davison Discovery Sciences, Deerfield, Illinois). The peptides were eluted via a 5–40% B gradient in 90 min into a nanoelectrospray-ionization LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, California). The mass spectrometer was operated in a data-dependent mode in which an initial MS scan recorded the mass range of #m/z# 500–2000, and then the six most abundant ions were selected automatically for collision-activated dissociation (CAD). The spray voltage was 3.0 kV. The normalized collision energy was set at 35% for MS-MS. All MS-MS data were searched against the Swissprot Mus musculus (mouse) protein database using the SEQUEST program. Precursor tolerance was set to 1.4 Da, and fragment tolerance was set to 1.0 Da. A static modification of 57.0215 Da for cysteine residues, and variable modifications of 15.9949 Da for methionine oxidation, 28.0302 Da for light-dimethylation, and 32.0553 Da for heavy-dimethylation on lysine and N-terminus were applied. One missed cleavage site of trypsin was allowed. Filtering criteria for the peptide identification were chosen so that the false discovery rate was lower than 2% determined by the reversed database search. These criteria included Xcorr ≥ 1.5 (+1 charge), 2.2 (+2 charge) and 3.0 (+3 charge); ΔCn ≥ 0.1; peptide probability ≤ 0.001. At least two peptides that met the previously mentioned criteria were required for protein assignment. Relative quantitation was achieved by the PepQuant function in the Bioworks software (Thermo Fisher Scientific, San Jose, California). The ratios of heavy- to light-labeled proteins were normalized according to the ratio obtained by the spiked chicken ovalbumin. Specifically, the raw data files were searched against a database composed only of chicken ovalbumin, and the identified heavy- and light-labeled peptides were compared by PepQuant and a 1.1:1 ratio for heavy-labeled to light-labeled peptide pairs were obtained. All the ratios of identified mouse proteins were then normalized to this internal standard ratio.  

The proteomics studies on serum samples usually suffer from the extreme dynamic range and complexity of proteins. By applying the workflow shown in Figure 1, we incorporated glycoprotein enrichment via lectin affinity chromatography and improved separation of peptides by 2D reversed phase LC before tandem MS experiments to achieve the characterization and quantitation of a total of 280 proteins. Among the proteins identified, 21 exhibited larger than twofold decrease and 28 exhibited larger than twofold increase in their concentrations in the infected samples.  

Glycoprotein enrichment by lectin affinity chromatography:

 The current bottleneck of discovering biomarkers in biofluid using MS is its limited dynamic range of detection compared with a much larger range of protein concentrations in the samples. Recent studies have shown that an effective way to cope with the complexity of the proteome is to use affinity chromatography methods to select a subproteome with a common type of modification. Lectins have been used in glycoprotein and glycopeptide isolation in proteomics, which is referred to as the "glyco-catch method" (6). In our study, to maximize the coverage of the glycoproteome, two lectins with broad selectivities were combined in a single column. Con A has a high affinity to high-mannose type N-glycans, whereas WGA is selective for N-acetyl-glucosamine (GlcNAc). The effectiveness of glycoprotein enrichment has been demonstrated by the identification of several glycoproteins with very low abundance, which will be discussed later in this section. The effectiveness is also evident from the depletion of many of the most abundant proteins. For example, serum albumin, by far the single most abundant protein in blood, takes up about 50% of the total protein mass and usually dominates the identified peptide list (7). However, with the lectin selection, albumin no longer appears on the top of the list. Instead, glycoproteins such as α-2-macroglobulin and complement C3 precursor were identified with the highest scores. It is worth noting that albumin was identified with a moderate number of peptides, due to the interactions with the glycoproteins it carries, or to the nonspecific interactions with the lectin proteins. To further deplete several of the most abundant proteins and enhance the detection of proteins in the low concentration region, an immunodepletion treatment before lectin selection will be desirable (7).  

Isotopic labeling by reductive amination:

 The isotopic labeling technique is based upon the dimethylation of peptide primary amine groups (lysine or N-terminus) with formaldehyde and deuterated formaldehyde in the presence of cyanoborohydride, as shown in Figure 2 (8). A mass difference of 4 Da is produced for each labeling site. The reaction is fast and easy to perform. In a preliminary study, to test the effectiveness and reproducibility of this quantitation approach, different amounts of a standard glycoprotein, chicken ovalbumin, at ratios of 1:1, 1:2, 1:5, and 1:10, were spiked into equal amount of mouse serum samples, respectively, and the quantitation was achieved by a quadrupole time-of-flight (QTOF) mass spectrometer. As expected, most of the mouse serum tryptic peptides appeared to be approximately 1:1 isotopic pairs in the spectra. As for the chicken ovalbumin, four tryptic peptides were identified and the light-to-heavy ratios were measured by integration of their peaks in the chromatogram. The results are shown in Table I. Although the quantitation method starts to lose its linearity above the ratio of 10:1, it is very accurate for a moderate change in ratio. Furthermore, a ratio larger than 10:1 itself would be an indication of a dramatic change in the protein profile, and other immuno-based quantitation methods can be employed to further validate the extent of protein expression change.  

Table I: Relative quantitation of four tryptic peptides from chicken ovalbumin			

Characterization and quantitation of low-abundance proteins:

 Figure 3a shows a representative example of MS-MS detection and identification of a tryptic peptide from apolipoprotein E (ApoE). ApoE is an O-glycosylated protein with a concentration of 50–80 μg/mL (9) in both human and mice serum. This protein was implicated previously in TSE disease progression, with an elevated level in the brains of scrapie-infected mice and has been detected in some prion protein deposits (10,11). In the same study, even lower abundance proteins such as epidermal growth factor receptor (EGFR), a membrane glycoprotein with a reported abundance as low as 400 ng/mL in serum samples (12), also were identified (Figure 3b). EGFR is a known transmembrane glycoprotein with three known glycosylation sites and seven potential glycosylation sites.  

Chicken ovalbumin is used as an internal standard because it is a glycoprotein that can be pulled down readily by Con A, and a separate database serach can be performed to distinguish it from mouse proteins. The heavy- to light-labeled ratios are calculated from the ratios of integrated areas of the peptides in the extracted ion chromatograms. All the tryptic peptides identified for the spiked chicken ovalbumin were averaged to give a 1.1:1 ratio for heavy-labeled to light-labeled peptide pairs, with only 10% error for such a multistep procedure. All of the relative quantitation information was normalized according to this ratio. Extracted ion chromatograms of two isotopically labeled pairs of peptides are shown in Figure 4. Both panels clearly show the co-elution relationship between the heavy- and light-labeled peptide pairs. Panel A shows a peptide assigned to complement factor H precursor with a 1.1 to 1 ratio. This is a secreted glycoprotein with five known glycosylation sites and two potential glycosylation sites. Panel B shows a tryptic peptide FVEEIIEETK from neurofilament medium polypeptide (neurofilament M) exhibiting about a sixfold increase in abundance. A significant peak above the baseline level was extracted for the heavy-labeled peptide, whereas only a small amount of signal was extracted for the light-labeled counterpart. The glycosylation sites of neurofilament M have been characterized by the combination of WGA weak affinity chromatography and MS equipped with electron-capture dissociation (ECD) (13). Neurofilament proteins previously have been shown to be associated with a number of neurodegenerative diseases (14). For example, it has been reported that the levels of antibodies against neurofilament M are elevated in multiple sclerosis (15). The characterization and relative quantitation of this protein of particular interest has demonstrated clearly that the application of our approach in discovering biomarkers in prion diseases is very promising.  

In summary, we have successfully developed an analytical platform that can detect and quantify glycoproteins readily in complex biofluid samples. By applying this platform, 280 proteins were identified and quantified, among which 49 proteins have shown larger than twofold changes in the blood samples from mice infected with prion disease. Although this list is preliminary and further replicate experiments must be done to validate the findings, this comparative glycoproteomic methodology offers significant promise in the search for low-abundance and glycosylated disease biomarkers.  

In future studies, immuno-depletion of the most abundant proteins before lectin selection will be desirable. Different lectin combinations with various selectivities towards glycan structures will generate different profiles of the serum glycoproteome and provides more information on the changes of glycoproteome of prion disease. Once a panel of proteins can be detected with reproducible changes by MS, other methods such as antibody-based assays will be required for validation before the candidates can be used for diagnostic purposes.  

The authors would like to thank the University of Wisconsin Human Proteomics Program for access to the LTQ instrument. This work was supported in part by National Institutes of Health through grant AI0272588, the Congressionally Directed Medical Research Program and National Prion Research Program, and the Wisconsin Alumni Research Foundation at the University of Wisconsin-Madison. L.L. acknowledges an Alfred P. Sloan Research Fellowship. 

Xin Wei*, Allen Herbst†, Joshua Schmidt‡, Judd Aiken†, and Lingjun Li*†

	* Department of Chemistry, University of Wisconsin-Madison 

				 †Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison  

				 ‡School of Pharmacy, University of Wisconsin-Madison, Madison 

Please direct correspondence to Lingjun Li at 

(1)   B. Caughey, R.E. Race, D. Ernst, M.J. Buchmeier, and B. Chesebro, 

(2)   D.R. Borchelt, M. Scott, A. Taraboulos, N. Stahl, and S.B. Prusiner, 

(3)   O. Schaller, R. Fatzer, M. Stack, J. Clark, W. Cooley, K. Biffiger, S. Egli, M. Doherr, M. Vandevelde, D. Heim, B. Oesch, and M. Moser, 

(7)   M. Ramstrom, C. Hagman, J.K. Mitchell, P.J. Derrick, P. Hakansson, and J. Bergquist, 

(8)   J.L. Hsu, S.Y. Huang, N.H. Chow, and S.H. Chen, 

(9)   A.H. Hasty, M.F. Linton, L.L. Swift, and S. Fazio, 

(10) J.F. Diedrich, P.E. Bendheim, Y.S. Kim, R.I. Carp, and A.T. Haase, 

(11) J.F. Diedrich, H. Minnigan, R.I. Carp, J.N. Whitaker, R. Race, W. Frey, and A.T. Haase, 

(12) A.T. Baron, J.M. Lafky, C.H. Boardman, S. Balasubramaniam, V.J. Suman, K.C. Podratz, and N.J. Maihle, 

(13) K. Vosseller, J.C. Trinidad, R.J. Chalkley, C.G. Specht, A. Thalhammer, A.J. Lynn, J.O. Snedecor, S. Guan, K.F. Medzihradszky, D.A. Maltby, R. Schoepfer, and A.L. Burlingame, 

(15) A. Bartos, L. Fialova, J. Soukupova, J. Kukal, I. Malbohan, and J. Pit'ha, 

The authors examine MS-based methods to search for protein biomarkers of prion diseases from plasma samples.

LCGC North America-02-01-2009

Testing Method Performance

The Early Days of HPLC at DuPont

Calendar

A Mass Spectrometry Primer, Part IV

Facilitating Discovery of Prion Disease Biomarkers by Quantitative Glycoproteomics

Short Courses

ICP MS Detection for HPLC Analyses of Pharmaceutical Products

Extraction and Preconcentration of Residues and Contaminants in Food Samples Using Membrane Techniques