iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer.
For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent-labeled samples. Although the popular LTQ linear ion trap supports analyzing iTRAQ reagent-labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent-labeled peptides, and increasing microscan acquisition and repeat count improves quantification but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found that results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent-labeled peptides, and provide guidelines for successful quantitative proteomic studies.
Griffin
Timothy J
TJ
Department of Biochemistry, Molecular Biology, and Biophysics, Center for Mass Spectrometry and Proteomics, University of Minnesota, Minneapolis, Minnesota 55455, USA. tgriffin@umn.edu
Xie
Hongwei
H
Bandhakavi
Sricharan
S
Popko
Jonathan
J
Mohan
Archana
A
Carlis
John V
JV
Higgins
LeeAnn
L
D002853:Chromatography, Liquid / Q000379:methods; D003627:Data Interpretation, Statistical; D004867:Equipment Design; D005656:Fungal Proteins / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007477:Ions; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*
D005656:Fungal Proteins; D007202:Indicators and Reagents; D007477:Ions; D010455:Peptides; D011506:Proteins; D020543:Proteome