The commercialization of high-pressure liquid chromatography (LC) systems, in combination with sub-2-Ám particle high performance
liquid chromatography (HPLC) columns, has enabled scientists to generate highly efficient LC–tandem mass spectrometry (MS-MS)
methods for separating peptides in complex matrices. The ability to reliably quantify a single peptide with baseline peak
widths of ~4.5 s is relatively simple; however, this becomes significantly more challenging when performing highly multiplexed
selected reaction monitoring (SRM) analyses. Long duty cycles involved in multiplexed analyses can severely impact the ability
to acquire sufficient data points over a peptide peak. The inclusion of time-resolved SRM functionalities into the mass spectrometer
control software has resolved this issue, where large numbers of peptides can now be quantified using short LC–MS-MS methods.
The benefits of time-resolved SRM analyses are discussed, along with examples of LC–MS-MS applications.
Mass spectrometry (MS), in combination with separation techniques such as gas chromatgraphy (GC)and liquid chromatography
(LC), has been used to quantify small molecules in biological matrices for decades. The application of electrospray ionization
(ESI) has enabled the same instrumentation to detect and analyse peptides and proteins in combination with LC. Protein analysis
is simplified through the addition of proteolytic enzymes, which cleave the protein at specific amino acid residues, converting
the protein into smaller, more manageable peptide fragments. The most commonly used protease is trypsin, which cuts the peptide
backbone at the C-terminal side of lysine (K) and arginine (R) residues (except immediately prior to proline [P]).
The enzymatic digestion of a protein can generate a large number of surrogate peptides, which significantly increases the
complexity of the matrix; however, it also enables analysts to monitor multiple peptides from the same protein. Furthermore,
other proteins present in the same matrix will also be digested into respective peptides, therefore enabling a multiplexed
protein analysis. Some laboratories have used LC–MS-MS and selected reaction monitoring (SRM)-based analysis to monitor significant
numbers of analytes, such as Anderson and Hunter (1), who used 137 SRM transitions to monitor 53 proteins in a single experiment.
This work involved the use of a nanobore LC system, which gave excellent sensitivity. However, each analysis had a run time
in excess of 40 min. For LC–MS-MS to be used routinely for protein analysis of clinical samples, the run time must be reduced
significantly. Using a 40-min run time will allow a throughput of 36 analyses per day; however, if a runtime of 4 min was
achieved, the throughput would increase to 360 analyses. This reduction in time has been demonstrated by using high-flow-rate
systems, with a concomitant drop in sensitivity that would be expected moving from nano to normal flow rates (2).
One additional drawback from moving to higher flow rates is that the multiplexing capability is severely reduced because of
the need to obtain in excess of 12 points over a chromatographic peak. This article describes advances in both LC and MS instrumentation
that have facilitated the development of highly multiplexed analyses that can be performed using short run times.
Ultrahigh-Pressure Liquid Chromatography
Recent developments in both LC instrumentation and stationary phase particle sizes have significantly improved the ability
to separate and detect peptides within digests of complex matrices such as plasma. The development of ultrahigh-pressure capable
systems has enabled the use of sub-2-Ám particles within LC columns. Using sub-2-Ám particle columns enables analysts to generate
peptide peak widths in the region of 1 s at half height, giving significant improvements in peptide resolution compared to
both normal high performance liquid chromatography (HPLC) separations, and nanobore separations (2). This peak sharpening
has resulted in increases in sensitivity over normal HPLC, as the peptides are eluted in a smaller volume in the electrospray
ionization (ESI) source.
The ability to generate sharp peptide peaks is not limited to sub-2-Ám particles, as high resolution peptide separations have
also been achieved using fused-core columns (3). Fused-core particles have an impermeable core with a diameter of 1.7 Ám and
a 0.5-Ám layer of porous solid phase material bonded to the surface of the particle. These columns generate almost identical
separation characteristics as the sub-2-Ám particle columns; however, the loadability of the column is lower because of the
reduction in solid-phase material available for performing separations.