Simultaneous quantification and identification using 18O labeling with an ion trap mass spectrometer and the analysis software application "ZoomQuant".
Stable isotope labeling with (18)O is a promising technique for obtaining both qualitative and quantitative information from a single differential protein expression experiment. The small 4 Da mass shift produced by incorporation of two molecules of (18)O, and the lack of available methods for automated quantification of large data sets has limited the use of this approach with electrospray ionization-ion trap (ESI-IT) mass spectrometers. In this paper, we describe a method of acquiring ESI-IT mass spectrometric data that provides accurate calculation of relative ratios of peptides that have been differentially labeled using(18)O. The method utilizes zoom scans to provide high resolution data. This allows for accurate calculation of (18)O/(16)O ratios for peptides even when as much as 50% of a (18)O labeled peptide is present as the singly labeled species. The use of zoom scan data also provides sufficient resolution for calculating accurate ratios for peptides of +3 and lower charge states. Sequence coverage is comparable to that obtained with data acquisition modes that use only MS and MS/MS scans. We have employed a newly developed analysis software tool, ZoomQuant, which allows for the automated analysis of large data sets. We show that the combination of zoom scan data acquisition and analysis using ZoomQuant provides calculation of isotopic ratios accurate to approximately 21%. This compares well with data produced from (18)O labeling experiments using time of flight (TOF) and Fourier transform-ion cyclotron resonance (FT-ICR) MS instruments.
Hicks
Wayne A
WA
National Proteomics Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53213, USA. whicks@mcw.edu
Halligan
Brian D
BD
Slyper
Ronit Y
RY
Twigger
Simon N
SN
Greene
Andrew S
AS
Olivier
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D006736:Horses; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis; D010103:Oxygen Isotopes; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000032:analysis; D011817:Rabbits; D051381:Rats; D015203:Reproducibility of Results; D012984:Software; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
D009211:Myoglobin; D010103:Oxygen Isotopes; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; C516610:Ptpn1 protein, rat