A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.
The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples.
Kuzyk
Michael A
MA
Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC, Canada.
Ohlund
Leanne B
LB
Elliott
Monica H
MH
Smith
Derek
D
Qian
Hong
H
Delaney
Allen
A
Hunter
Christie L
CL
Borchers
Christoph H
CH
D002853:Chromatography, Liquid / Q000379:methods; D029968:Escherichia coli Proteins / Q000032:analysis; D010455:Peptides / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D029968:Escherichia coli Proteins; D010455:Peptides