Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations.
Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.
Elias
Joshua E
JE
Department of Cell Biology, 240 Longwood Ave., Harvard Medical School, Boston, Massachusetts 02115, USA.
Haas
Wilhelm
W
Faherty
Brendan K
BK
Gygi
Steven P
SP
D000465:Algorithms; D019985:Benchmarking; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D013673:Technology Assessment, Biomedical
D020543:Proteome