title: Normalization approaches for removing systematic biases associated with mass spectrometry and label-free proteomics.
abstract: Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias to some degree, assigned ranks among the techniques revealed that for most LC-FTICR-MS analyses linear regression normalization ranked either first or second. However, the lack of a definitive trend among the techniques suggested the need for additional investigation into adapting normalization approaches for label-free proteomics. Nevertheless, this study serves as an important step for evaluating approaches that address systematic biases related to relative quantification and label-free proteomics.
authors.affiliation: Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Callister
authors.forename: Richard C
authors.initials: RC
authors.lastname: Barry
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: Ethan T
authors.initials: ET
authors.lastname: Johnson
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
authors.forename: Bobbie-Jo M
authors.initials: BJ
authors.lastname: Webb-Robertson
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Mary S
authors.initials: MS
authors.lastname: Lipton
mesh_terms: D000818:Animals; D015982:Bias; D000697:Central Nervous System Stimulants; D003342:Corpus Striatum / Q000187:drug effects / Q000378:metabolism*; D034301:Deinococcus / Q000378:metabolism; D005583:Fourier Analysis; D013058:Mass Spectrometry; D008694:Methamphetamine / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000032:analysis*; D040901:Proteomics
chemical_list: D000697:Central Nervous System Stimulants; D010455:Peptides; D008694:Methamphetamine