An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags.
Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Stephanie M
SM
Cologna
Biomedical Mass Spectrometry Facility, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Christopher A
CA
Crutchfield
Proteome Software, Inc. , 1340 SW Bertha Boulevard, Portland, Oregon 97219, United States.
Brian C
BC
Searle
Section on Membrane and Cellular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Paul S
PS
Blank
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Cynthia L
CL
Toth
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Alexa M
AM
Ely
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Jaqueline A
JA
Picache
Biomedical Mass Spectrometry Facility, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Peter S
PS
Backlund
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Christopher A
CA
Wassif
Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Forbes D
FD
Porter
Biomedical Mass Spectrometry Facility, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS , 31 Center Drive, Bethesda, Maryland 20892, United States.
Alfred L
AL
Yergey
D045424:Complex Mixtures / Q000737:chemistry*; D056945:Hep G2 Cells; D006801:Humans; D007477:Ions / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D059748:Proteolysis; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D045424:Complex Mixtures; D007477:Ions; D010455:Peptides; D020543:Proteome; D014357:Trypsin
MALDI-MS; fold-change; iTRAQ; quality control; quantitative proteomics; time-of-flight