Reference map for liquid chromatography-mass spectrometry-based quantitative proteomics.
The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC-tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.
Kim
Yeoun Jin
YJ
Celera, Rockville, MD 20850, USA.
Feild
Brian
B
Fitzhugh
William
W
Heidbrink
Jenny L
JL
Duff
James W
JW
Heil
Jeremy
J
Ruben
Steven M
SM
He
Tao
T
D000476:Alkanesulfonates; D001943:Breast Neoplasms / Q000737:chemistry; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D003626:Data Display; D030562:Databases, Protein; D005260:Female; D006801:Humans; D010455:Peptides; D020543:Proteome / Q000032:analysis* / Q000592:standards; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012685:Sepharose / Q000031:analogs & derivatives; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000476:Alkanesulfonates; D010455:Peptides; D020543:Proteome; C057181:poly(2-sulfoethyl aspartamide)silica; C035872:thiopropyl-sepharose; D012685:Sepharose