Liquid-liquid extraction (LLE) and solid-support liquid-liquid extraction (SSLE) are common sample preparation choices in regulated bioanalysis. LLE and SSLE can generate high analyte recoveries, clean extracts, and LLE is perceived as low cost. Depending on the compounds analyzed, however, these methods must often be optimized. Extraction solvents may need to be acidified, basified, buffered or low percentages of more polar solvents may be required to simultaneously achieve high recoveries for metabolites and related compounds, as well as the primary analyte. In many cases, the choice of solvent may lead to extracts particularly saturated with phospholipids (PLs). As well as contributing to matrix effects, residual PLs can build-up on the analytical column and the LC system. PL build-up may cause analyte signal variability, suppression of MS response, and potentially lead to instrument down-time. As an alternative to LLE and SSLE, the Ostro sample preparation plate provides a simple, one-step method which achieves high recoveries for diverse analyte types without optimization. The Ostro plate also removes more PLs than LLE and more or equivalent PLs than SSLE. In addition, as Ostro extraction is performed with formic acid in acetonitrile, samples can be diluted and directly injected. This is in contrast to LLE and SSLE extracts which must be evaporated and reconstituted prior to injection. 

 Figure 1: Comparison of PLs remaining in Ostro samples extracted using the basic method and (a) LLE using a variety of extraction solvents: (LLE 1) 100% MTBE (LLE 2) 98/2% hexane/isoamyl alcohol (LLE 3) 60/40% cyclohexane/dichlormethane and (b) SSLE with the same solvents. 

A diverse set of analyte samples were processed on the Ostro 96-well plate using a generic, simple protocol provided by the manufacturer. Using the generic protocol, the Ostro plate provided overall significantly better recovery for the analytes tested than LLE [Figure 1(a)] and SSLE [Figure 1(b)] which were each tested with 3 extraction solvents: 100% MTBE, 98/2% hexane/isoamyl alcohol (v/v), and 60/40% cyclohexane/dichloromethane (v/v). The average recovery for all analytes using the Ostro plate was 80%. The average recovery for all analytes using LLE and SSLE was 23% and 17%. The Ostro 96-well plate also removed significantly more PLs than LLE [Figure 2(a)] and removed significantly less or a comparable level of PLs to SSLE [Figure 2(b)] In addition, matrix effects were calculated. The Ostro plate extracts generated less or equivalent matrix effects than LLE and SSLE.   

Figure 2: Average analyte recovery of a diverse analyte mix using the Ostro 96-well plate and (a) LLE with 100% MTBE, 98/2% hexane/isoamyl alcohol (v/v), and 60/40% cyclohexane/dichloromethane and (b) SSLE with the same solvents. 

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Articles

Pharmaceutical/Drug Discovery

Providing a Universal, One-step Alternative to Liquid-Liquid Extraction in Bioanalysis

This application note describes the extraction of a range of benzodiazepines from human urine using ISOLUTE SLE+ supported liquid extraction plates (96-well) and columns prior to LC–MS/MS analysis.  Supported liquid extraction is an efficient and cost-effective alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation and significantly reduced sample preparation time. 

This application note outlines the procedure using both the ISOLUTE SLE + 1 mL sample volume column (part number 820-0140-C) optimized for a 1 mL pretreated urine sample volume and the ISOLUTE SLE+ 96-well extraction plate (part number 820-0200-P01) optimized for a 200 µL pre-treated sample. Method parameters and dilution factors have been optimized to maximize recoveries and minimize ion suppression. 

 Table 1: MRM transitions in positive mode and detected LOQs for a range of benzodiazepines. 

Sample pre-treatment: To 1 mL of urine add 100 mM  ammonium acetate pH 5 (950 µL) and  -glucuronidase (50 µL)  enzyme (~4500 u/mL). Hydrolyze  in a water bath at 60 °C for 2 h. 50 µL of enzyme is required per  mL of urine used.  For a 200 µL sample use ISOLUTE SLE+ 200 Supported  Liquid Extraction plate, part  number 820-0200-P01. 

Sample load:  Load pre-treated sample (200 µL)  to plate followed by a pulse  of vacuum to initiate flow and  leave for five min to  completely adsorb. 

Analyte elution: Elute with dichloromethane (1mL).  Leave to flow under gravity for five  min, then apply short pulse of  vacuum. 

Post extraction: Evaporate to dryness at room  temperature (80 L/min) and  reconstitute in HPLC grade  water/acetonitrile (70/30,v/v)  (200 µL). Cap with sealing mat  and vortex gently (60 s). 

 For a 1 mL sample use ISOLUTE  SLE + 1 mL sample volume  column, part number  8200140C. 

Sample load: Load pre-treated sample (1 mL) to  plate followed by a pulse of  vacuum to initiate flow and leave  for five min to completely adsorb. 

Analyte elution: Elute with dichloromethane (2.5 mL). Leave to flow under gravity for  five min, then follow with a further  aliquot of dichloromethane (2.5 mL),  apply a short pulse of vacuum. 

Post extraction: Evaporate to dryness at room  temperature (80 L/min) and  reconstitute in HPLC-grade  water/acetonitrile (70/30, v/v) (1 mL). Vortex gently (60 s). 

Instrument:      Waters Alliance 2795 

Column:      Phenomenex Kinetex C18 100 Å  column (2.6 µm, 50 x 2.1 mm id) 

Mobile phase:      0.1% formic acid (aq)/acetonitrile  (70/30, v/v) (isocratic) 

Injection:      25 µL (partial loop) 

Instrument:  Waters Ultima Pt triple quadrupole  mass spectrometer equipped with  an electrospray interfacefor mass  analysis 

Extracted samples demonstrated consistent recoveries between 85-110% with RSDs below 10%. Lowest LOQ demonstrated was 100 pg/mL for at least four analytes and this could be improved with more sensitive mass spectrometry. 

Figure 1: Average analyte recoveries of benzodiazepines analytes on 96 fixed well plates at 10 ng/mL (n=7). 

This method demonstrates a robust and effective extraction of a full range of benzodiazepines from the challenging biological matrix of human urine with hydrolysis. The hydrolysis step shown here used the enzyme helix pomatia but other enzymes can be used with no loss of efficacy of the application.

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Medical/Biological

Extraction of Benzodiazepines From Human Urine Using ISOLUTE SLE+ 96-Well Plates and Columns prior to LC–MS/MS Analysis

There are various workflows for LC–MS-based protein bioanalysis. However, certain primary steps are often common to all workflows. These consist of some or all of the following: identification of unique signature peptides from the protein, isolation of the protein from a biological matrix (such as plasma), trypsin digestion of the protein or of the entire matrix containing the protein (if pre-fractionation was not performed), isolation/clean-up of signature peptides from the digest, and MRM quantitation of peptides using triple quadrupole MS. 

Unless very selective enrichment of the protein is performed prior to digestion, the complexity of the digested sample, matrix effects, and the presence of isobaric interferences can make low-level quantitation difficult. The use of mixed-mode SPE to isolate signature peptides from a digest reaction can significantly improve the quality of the quantitative data. Residual denaturants, reduction, alkylation, and digest reagents will be removed during this process, as well as peptides that are more or less polar than the peptides of interest, markedly decreasing the complexity of the sample. 

In this work, the monoclonal antibody, trastuzumab, a breast cancer drug, was used to demonstrate the effectiveness of an SPE clean up. Trastuzumab was spiked into serum, and the entire serum sample (without additional pre-fractionation) was denatured, reduced, alkylated and digested with trypsin overnight. Digest samples were analysed with and without additional clean up using mixedmode SPE to highlight the benefits of this important step in a protein bioanalysis workflow. 

Oasis MCX µElution 96-well plate (part#186001830BA) 400—750 mL of diluted serum digest (200—375 mL digest, 200—375 mL 4% H3PO4 in water) was loaded onto the plate. 

The sample wells were washed with 2% formic acid and then 5% methanol. The peptides were eluted twice with 25 µL of 40/5/55% ACN/conc. NH4OH/water (v/v/v). 

Unique trastuzumab peptides were first identified using an i

 approach. Several of the most sensitive of these peptides were chosen to represent trastuzumab in a quantitative LC–MS–MS assay. During analysis, it was noted that residual digest reagents and closely related interferences impacted LC–MS–MS sensitivity and robustness. For example, sensitivity for a system test standard decreased over time if digest samples were directly injected. In addition, many peaks were present in the MRM trace for the peptide of interest [Figure 1(a)] and the peptide signal in the presence of digested matrix was significantly lower than that of a digested solvent standard (data not shown). 

 Figure 1: A signature peptide DTYIHWVR detected on a Xevo TQ-S before (a) and after (b) SPE sample cleanup. 

A mixed-mode SPE method was developed to clean up digest samples and to isolate the signature peptides. The SPE method not only reduced background noise, simplified the MRM spectrum and eliminated isobaric interferences, but also substantially increased signal for the signature peptide [Figure 1(b)]. SPE recovery for the signature peptide was 83%. The use of µElution format SPE enables samples to be concentrated without evaporation due to the high loading capacity and low elution volumes required.  

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Clean-up of Plasma and Serum Protein Digests for Protein Bioanalysis

 Nowadays, there is an increasing demand to automate sample pretreatment in order to reduce labour costs and increase productivity. In environmental laboratories trace enrichment is one of the most laborious tasks, as this involves activities such as liquid-liquid extraction (LLE), clean-up and re-concentration by solvent evaporation. In many cases, solid phase extraction (SPE) has successfully replaced LLE. 

 Figure 1: Chromatogram of PAH standard at 1 Âµg/L. 

The use of online SPE requires less solvents and brings an additional benefit: there is no need to evaporate the extraction solvent, which is beneficial for overall recoveries of the target compounds. 

Moreover, all of the desired compounds will be detected. In case of off-line pretreatment an aliquot of the obtained extract is always injected into the analysis system. 

Another development in sample analysis is increasing throughput. In many cases, speed of analysis is the key factor. In environmental analysis, however, samples are quite complex. Even where compounds are detected using more selective detection methods (for example, fluorescence detection), enough separation power is still necessary to fully resolve all detected compounds. Otherwise false positive results may occur.  

Table 1: Table with wavelength settings. 

In some cases, the possibility of false positives can be ruled out or, at least, diminished by the use of a second detector. Sometimes a fast analysis method is used for screening. When a positive result emerges it needs to be confirmed by a second technique. As stated above in environmental analysis, samples are very complex so only methods with very selective detection techniques (for example LC/MS/MS or GC/MS) can be used for screening. 

Online SPE Method for the Determination of PAHs

Shimadzu Benelux, together with Vitens, the largest drinking water producer in the Netherlands, has developed an online solid phase extraction (SPE) method for the determination of polycyclic aromatic hydrocarbons (PAH) in drinking water, ground water and surface water. The company supplies about 350 million m

 of drinking water annually to one third of the Netherlands and is internationally active in helping local drinking water companies in Africa, South America and Asia to improve their water supply and sanitation. 

Figure 2: Schematic representation of SPE system. 

The analysis of polycyclic hydrocarbons is primarily carried out using HPLC with UV, fluorescence detection or GC/MS, although the latter two are generally more accepted. The objective in this case was to combine SPE with an HPLC method with fluorescence detection. 

As a starting point an HPLC method for the complete separation of 16 EPA priority PAHs was taken.

  The method incorporates a C18 reversed-phase column onto which a water-acetonitrile binary high pressure gradient was executed. Separation was further improved by lowering the column temperature to 20 °C and adjusting the steepness of the gradient from 25 to 100 % acetonitrile in 30 min. The minimum resolution between the critical pair (acenaphtylene and fluorene) was around 3.4. The low temperature of the column gives maximum resolution for the PAH compounds, which enables easy switching of excitation and emission wavelengths of the RF-20Axs fluorescence detector. 

When method robustness was established, the HPLC system was further extended with online SPE capability. For this, an SPE cartridge, a pump with four solvent selection valves for cleaning, conditioning and flushing of the C8 SPE material and two 6-port 2-position valves were added. In this stage, more degassing capacity was added and the autosampler was replaced by a preparative SIL-10AP which is most suited for large volume injections. 

Before sample loading, the SPE cartridge was first cleaned with acetonitrile, conditioned with methanol and flushed with water. 4 mL of sample

  was injected and transported to the SPE cartridge with a flow of 1 mL/min of an acetonitrile-water mixture. PAH compounds tend to stick anywhere in the HPLC system so a solution of 33% acetonitrile is needed to overcome this problem. After sample loading, the cartridge was again flushed with water before the compounds were desorbed with 100% acetonitrile. The flow coming from the SPE cartridge was downstream mixed with water to form the wateracetonitrile gradient for elution of the PAH from the analytical column. 

Column:      Pursuit PAH3 4.6 mm i.d. × 100 mm L, 3 µm

Pre-column:      ChromSep Guard, Pursuit PAH 3 ×10 mm L

Mobile phase:      A: water/B: acetonitrile

Gradient:      B: 25 % –> 100 % (30 min)

Detection:      fluorescence (see table)

SPE column:      HySpere Resin SH 25, 2 × 10 mmL

Loading flow:      1 mL/min water-acetonitrile 7/3 (v/v) 

The limit of quantification (LOQ) lies between 0.0007 and 0.0059 µg/L depending on the matrix, except for naphthalene (0.0087 - 0.021 µg/L). The LOQ is calculated as three times the standard deviation of an addition of 0.01 µg/L which was determined on eight separate days. The repeatability is between 0.39 and 6.4 % (n = 8), the reproducibility ranges from 1.3 to 9.9 % (n = 8). 

Using the RF-20Axs, which is the most sensitive HPLC fluorescence detector in the world, satisfies the needs particularly for high-sensitivity environmental analysis. 

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1. All 16 EPA PAHs can be detected with a UV detector. With fluorescence detection only 15 out of 16 EPA PAHs could be detected, as acenaphthylene does not exhibit native fluorescence. 

2. Sample was mixed with acetonitrile in the sample vial prior to injection. 

Environmental

Tracing PAH Analysis with an Automated Sample Pretreatment Online SPE Method

Cellulose acetate (CA) is one of the most important cellulose derivatives. Its main applications are their use in membranes, films, fibres, plastics and filters. The chemical and physical properties of these derivatives are strongly influenced by the degree of substitution (DS) and the molar mass and molar mass distribution. Therefore, there is a strong need to characterize these properties.

SEC provides a convenient way for the characterization of molar masses and molar mass distribution of synthetic and natural polymers. However, a crucial balance of the polarities of the analyte, the solvent and the stationary phase is required for a reliable SEC method. Since DS of cellulose derivatives alters the polarity, solubility and aggregation behaviour, the SEC methods found in literature are usually valid only within a narrow DS-range. Published SEC methods usually apply THF or DMF modified by the addition of LiBr as eluents on styrene-divinylbenzene (SDV) columns. However, often neither the degree of substitution nor the DS-range applicable for the given chromatographic conditions are specified. The only SEC system suitable for cellulose acetate over a wide DS-range (DS range 0.4-1.2) uses pyridine/water on a GRAMS column. This application note reports on SEC-separations of high molar mass CAs for CAs of higher DS in the range DS = 1.5-3. 

Most samples were prepared by partial saponification of a commercial CA sample with a DS of 2.6. Other samples were of industrial origin. A Tosoh Biosystems EcoSECSystem (Tosoh, Stuttgart, Germany) equipped with a MALLS detector (DAWN DSP, Wyatt Technology, Santa Barbara, USA) was used. The stationary phase was a GRAM linear XL column (10 µm particle size, 30 × 0.8 cm, PSS Polymer Standards Service, Mainz, Germany). DMSO was used as the eluent. The low refractive index increment of cellulose acetates in DMSO (0.024-0.044 mL/g, depending on DS) does not allow for reliable molar mass determination by SEC-MALLS. The very sensitive EcoSEC RI, however, could easily detect those samples reliably. However, the light scattering instrument revealed aggregation if no or only low LiCl concentrations were used. Aggregation was not observed at LiCl concentrations above 50 mmol/L. Some of the samples required higher salt concentrations for dissolution. 

Figure 1 shows a comparison of the chromatograms of three CAs of different DS, which were prepared from the same base material. The elution volumes and the peak shapes of the samples are nearly identical, indicating that no degradation occurred during saponification. This has also been confirmed by molar mass determination using light scattering in a different solvent with higher sample contrast (dn/dc). The close agreement in the chromatograms of the samples can therefore be regarded as an indication of nearly identical calibration curves of the cellulose acetates despite differences in DS. The fourth sample of DS = 1.72 is a commercial sample. It is presented in order to show that the close agreement of the chromatograms is not due to an insufficient chromatographic resolution. The independence of the calibration curves on DS is advantageous. It will allow reliable molar mass determination of cellulose acetates without worrying about variations of SEC elution volume due to differences in DS.  

 Figure 1: Stacked overlay of chromatograms of cellulose acetates of different DS: DS (grey: DS = 2.45, red: DS = 2.09, green: DS = 1.59) derived from the same base material by partial saponification and a commercial sample (blue: DS = 1.72). 

General

SEC Method for Cellulose Acetates of Wide DS-Range

The introduction of single site catalysts and multiple reactor technology allows the design of new polyolefin resins, and the Chemical Composition Distribution (CCD) has become the most discriminating microstructure parameter in these polymers. The proper analysis of the CCD necessarily requires a fractionation process, typically by crystallizability; existing analytical techniques like Temperature Rising Elution Fractionation TREF

 and Crystallization Analysis Fractionation CRYSTAF

have shown in the last few years to be of great value in the polyolefins industry. Although analytical time has been reduced to a few hours per sample in both CRYSTAF and TREF, a faster method without sacrificing resolution was required, which ended in the development of a new technique known as Crystallization Elution Fractionation (CEF)

CEF was introduced in 2006 at the 1st International Conference on Polyolefin Characterization (ICPC) in Houston, TX

. CEF combines the separation power of CRYSTAF in the crystallization step with the separation achieved in TREF during the elution cycle. CEF has been shown to be superior to both TREF and CRYSTAF and is capable to provide very fast analysis of the composition distribution of polyolefins for high-throughput applications

Crystallization Elution Fractionation mechanism

The principles of TREF have been widely discussed

 and are shown schematically in Figure 1(a) where three components loaded on the head of the column are deposited 

(crystallization step) with no physical separation but acquiring the potential to be separated in the elution step.  

 Figure 1: Principles of TREF, DC and CEF. 

In our search for new techniques to improve resolution it was our intention to combine the separation power of TREF in the elution cycle with the CRYSTAF separation in the crystallization step, but CRYSTAF as such does not physically separate fractions, it only monitors the crystallization process

. To achieve a physical separation of fractions in the crystallization step a new approach was developed that is defined as Dynamic Crystallization

In Dynamic Crystallization (DC) a small flow of solvent, F

, is passed through the column during cooling, in such a way that when a component reaches its crystallization temperature it is segregated from the moving solution and is anchored on the support while the other components, remaining in solution, will move along the column until they reach their own crystallization temperature. At the end of the crystallization cycle the components are separated inside the column according to crystallizability as shown in Figure 1(b). 

Once the cooling cycle is completed, the flow is interrupted and the column heated for a few minutes at a temperature, T

, where all components would be dissolved while remaining in the column; if applying a solvent flow, F

, at this step to elute the components out of the column, they will be separated according to CRYSTAF mechanism.  

Once Dynamic Crystallization was developed, it was easy to realize the possibility of combining this crystallization step with a final elution cycle, as in TREF, to obtain a new and extended separation. This is represented in Figure 1(c) where the DC cycle is followed by the temperature rising elution cycle, with a flow Fe as in TREF. The new process is known as Crystallization Elution Fractionation (CEF) because it combines the separation obtained in the crystallization step with the one obtained in the elution cycle; this is schematically represented in Figure 1(c) by the extended separation of the three components at the exit of the column in CEF analysis as compared to the TREF approach. 

CEF, DC and TREF experiments were performed in a specially developed benchtop instrument. As the interest of CEF technology is focused not only on classical Chemical Composition Distribution measurement but also on high throughput analysis, the instrument was built incorporating an autosampler with capacity of 70 vials of 10 ml.  

The CEF instrument diagram is quite simple, as shown in Figure 2. The autosampler dissolves the sample in o-dichlorobenzene and it is loaded into the injection valve loop through the syringe dispenser. The sample is injected with the pump flow into the column head and the DC process begins at a given cooling rate and crystallization flow. As the crystallization ends, the oven starts the heating programme and flow is adapted to the elution flow to obtain the CEF analysis passing through the column to a dual wavelength infrared detector, so concentration and composition can be measured at once. A dual capillary viscometer can be added to the system to measure the composition – molar mass dependence.  

Figure 2: Schematic diagram of the Polymer Char CEF bench-top instrument. 

CEF and TREF analyses of a blend of two metallocene resins were performed at the same cooling rate of 2 °C/min and with a heating rate of 2°C/min. The results are shown in Figure 3. 

The extended separation between peaks in CEF over TREF (8 °C) corresponds to the extra DC separation (present in CEF only), that at a heating rate of 2 °C/min results in 4 min separation time, which at an elution flow of 1 mL/min corresponds to an extended volume separation of 4 mL, indicating that the DC process deposited the fractions (coloured spots in Figure 3) along the column empty volume, separated 4 mL one from the other. 

Figure 3: Comparison of CEF and TREF analysis of a blend under similar analytical conditions. 

Resolution in CEF is quite superior to TREF, as shown by the baseline between peaks being closer to zero in CEF. 

The CEF analysis can be advantageous over TREF to improve resolution, as in Figure 3, or to speed-up analysis for highthroughput operation, achieving the same resolution power but at a significant shorter time than TREF. 

The use of a two heated zones autosampler, where samples are being prepared (automated vial filling and dissolution) to be injected into the CEF column, in combination with high-speed column technology, allows the analysis of samples every 25 min or 50 samples per day, at a crystallization rate of 5 °C/m and heating rate of 10 °C/m. 

The reproducibility of the technique is extremely good as shown in Figure 4 with 10 consecutive analyses of the same sample placed in different vials.  

Figure 4: Analysis of 10 samples in different vials of a multi-reactor resin. Analysis time per sample 25 minutes. 

A new approach to measure the Chemical Composition Distribution in polyolefins has been developed – Crystallization Elution Fractionation (CEF) – which is superior in resolution, speed and hardware simplicity over existing techniques. 

CEF is well adapted to high-throughput analysis in combinatorial chemistry tools, being capable of analysing samples with reasonable resolution in less than 30 min. 

It is expected that CEF will replace most of TREF and CRYSTAF work in the future analysis of the CCD in polyolefins; TREF and CRYSTAF instrumentation will be required only for the analysis of Polypropylene / Polyethylene combinations where under-cooling differences will demand it

Gustave Eiffel 8, Paterna, Valencia E-46980 Spain

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3. B. Monrabal, Crystallization Analysis fractionation, US Patent 

, John Wiley & Sons, 2000, p. 8074–8094.  

5. B. Monrabal, Crystallization Elution Fractionation, Patent Application WO 2007/104804 A2. 

 71–79  B. Monrabal, J. Sancho-Tello, N. Mayo, L. Romero (2007). 

 14-24, B. Monrabal, L. Romero, N. Mayo, J. Sancho-Tello (2009). 

8. Analytical and Bioanalytical Chemistry, 

(4), 1557 – 1561. B. Monrabal, P. del Hierro (2011). 

Polymers/Polymer Analysis

Crystallization Elution Fractionation: A New Approach to Measure the Chemical Composition Distribution in Polyolefins

Protein phosphorylation is an essential mechanism in eukaryotic cells to regulate vital cellular processes such as cell cycle or growth and differentiation. Altered regulation may result in critical diseases like cancer. Detailed characterization is therefore essential. Phosphoproteins are challenging samples for direct analysis using mass spectrometry (MS). The instrument must be able to characterize the molecules against a high background of abundant proteins in the sample. 

The new amaZon speed ETD ion trap MS is ready for this task. The instrument delivers a wide dynamic range, high spectral duty cycle and offers electron-transfer dissociation (ETD) fragmentation along with collision-induced dissociation (CID). By combining ETD with CID, the instrument is state-of-the-art in the characterization of phosphylation sites in proteins. 

  analysed human Raji B cell lysates. In brief, cells were digested and phosphopeptide purification was performed. Vacuum-dried peptide samples were then separated using an Ultimate 3000 nano-LC system (Dionex, Sunnyvale, CA, USA) coupled to an amaZon speed ETD ion trap equipped with CaptiveSpray ion source (both Bruker, Bremen, Germany). Scan speed was 8,100 u/s in MS and 52,000 in MS/MS. Acquisition was performed with combined CID and ETD fragmentation. All results were stored and analysed with ProteinScape (Bruker).  

In three technical replicates, an average of 320 proteins were identified. 313 proteins were identified as phosphoproteins. ProteinScape substantially simplified data analysis while CID in combination with ETD efficiently enabled mapping of phosphorylation sites on the majority of peptides studied. In [Figure 1(a)], phosphorylation of a peptide is identified using CID. ETD analysis is automatically triggered from the CID result. ETD fragmentation cleaves the peptide backbone, leaving the phosphate group in place and thus allows evaluation of the localization of the phosphate group [Figure 1 (b) and (c)] – in this case serine 3.  

 Figure 1: Characterization of phosphoproteins with CID and ETD: (a) CID identifies phosphorylated proteins, while ETD enables the location of the phosphate groups to amino acids (b and c). Here, serine 3 was identified as the phorsphorylation site.			

ETD triggered by CID is by far the most efficient technology to characterize post-translational modifications. The experiments cited here clearly demonstrate the superior analytical power of the combination of state-of-the-art ion trap technology with optimized fragmentation techniques and leading ion source design.  Powerful bioinformatics with ProteinScape enable comprehensive and reliable achievement of results.  

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, Phosphoproteome mapping with the amaZon speed ETD. (