(218f) Optimization of Chip-Based Infusion Nanoelectrospray Tandem Mass Spectrometry for the Rapid Analysis of Complex Proteomes

In
high-throughput proteomics, accuracy, coverage and analysis time are crucial
requirements. Sample introduction via automated nanoelectrospray infusion has
definite advantages when compared to liquid chromatographic (LC)-electrospray
ionization ESI sample delivery, including high-throughput operation,
automation, no carry-over or cross-contamination, and a constant sample matrix.
We have evaluated and optimized the use of a chip-based nanoelectrospray device
(Advion Nanomate) interfaced to a hybrid quadrupole time of flight mass
spectrometer (Applied Biosystems / MDS Sciex QStarXL) for the analysis of
tryptically digested samples: cytochrome C and a six-protein mixture
(containing cytochrome
C, lysozyme, alcohol dehydrogenase, bovine serum albumin, apo-transferrin, and b-galactosidase) at
concentrations from 5 to 500 fmol/µl. We
quantified the effects on the identification and peptide coverage of MS and
MS/MS scan times, Zip-Tip C₁₈ sample clean-up, in-gel digestion
residues and sample concentrations. Different survey (1-10 seconds) and MS/MS
(1-10 seconds) scan times were evaluated, and spectra were searched with Mascot
against generic and species specific databases. Each infusion experiment
consisted of 22 cycles in the mass spectrometer, and each cycle consisted of 1
survey and 8 MS/MS scans. Infusion results were compared to those from nanoflow
LC-ESI-MS/MS, which was run isocratically to maintain a quasi-uniform sample
introduction.

The results from the isocratic LC-ESI-MS/MS suggest
that the chip-based infusion yields greater coverage, with marginally lower
MOWSE scores. The optimal MS method, selected to take advantage of the extended
infusion without losing the high-throughput requirement, includes a 10 second
survey scan and 8 cycles of 3 second MS/MS scans. Each isocratic LC experiment,
from injection to injection, took approximately twice as long as each
chip-based experiment. We discuss the
feasibility of extending this methodology to more complex proteomes, such as
those obtained from the model cyanobacterium, Synechocystis sp. PCC6803,
as we have extensive experience with this organism [1]. Furthermore, we discuss
the role of this configuration in aiding identification of post-translational
modifications.

[1]
Gan CS, Reardon KF, Wright PC, Comparison of protein and peptide
prefractionation methods for the shotgun proteomic analysis of Synechocystis
sp. PCC 6803, Proteomics, 5(9), 2005, 2468-2478.