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Improved normalization of systematic biases affecting ion current measurements in label-free proteomics data.

ABSTRACT: Normalization is an important step in the analysis of quantitative proteomics data. If this step is ignored, systematic biases can lead to incorrect assumptions about regulation. Most statistical procedures for normalizing proteomics data have been borrowed from genomics where their development has focused on the removal of so-called 'batch effects.' In general, a typical normalization step in proteomics works under the assumption that most peptides/proteins do not change; scaling is then used to give a median log-ratio of 0. The focus of this work was to identify other factors, derived from knowledge of the variables in proteomics, which might be used to improve normalization. Here we have examined the multi-laboratory data sets from Phase I of the NCI's CPTAC program. Surprisingly, the most important bias variables affecting peptide intensities within labs were retention time and charge state. The magnitude of these observations was exaggerated in samples of unequal concentrations or "spike-in" levels, presumably because the average precursor charge for peptides with higher charge state potentials is lower at higher relative sample concentrations. These effects are consistent with reduced protonation during electrospray and demonstrate that the physical properties of the peptides themselves can serve as good reporters of systematic biases. Between labs, retention time, precursor m/z, and peptide length were most commonly the top-ranked bias variables, over the standardly used average intensity (A). A larger set of variables was then used to develop a stepwise normalization procedure. This statistical model was found to perform as well or better on the CPTAC mock biomarker data than other commonly used methods. Furthermore, the method described here does not require a priori knowledge of the systematic biases in a given data set. These improvements can be attributed to the inclusion of variables other than average intensity during normalization.

SUBMITTER: Rudnick PA

PROVIDER: S-EPMC4014289 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Improved normalization of systematic biases affecting ion current measurements in label-free proteomics data.

Rudnick Paul A PA Wang Xia X Yan Xinjian X Sedransk Nell N Stein Stephen E SE

Molecular & cellular proteomics : MCP 20140221 5

Normalization is an important step in the analysis of quantitative proteomics data. If this step is ignored, systematic biases can lead to incorrect assumptions about regulation. Most statistical procedures for normalizing proteomics data have been borrowed from genomics where their development has focused on the removal of so-called 'batch effects.' In general, a typical normalization step in proteomics works under the assumption that most peptides/proteins do not change; scaling is then used t ...[more]

PMID: 24563535

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Project description:BackgroundLabel-free quantitative proteomics holds a great deal of promise for the future study of both medicine and biology. However, the data generated is extremely intricate in its correlation structure, and its proper analysis is complex. There are issues with missing identifications. There are high levels of correlation between many, but not all, of the peptides derived from the same protein. Additionally, there may be systematic shifts in the sensitivity of the machine between experiments or even through time within the duration of a single experiment.ResultsWe describe a hierarchical model for analyzing unbiased, label-free proteomics data which utilizes the covariance of peptide expression across samples as well as MS/MS-based identifications to group peptides-a strategy we call metaprotein expression modeling. Our metaprotein model acknowledges the possibility of misidentifications, post-translational modifications and systematic differences between samples due to changes in instrument sensitivity or differences in total protein concentration. In addition, our approach allows us to validate findings from unbiased, label-free proteomics experiments with further unbiased, label-free proteomics experiments. Finally, we demonstrate the clinical/translational utility of the model for building predictors capable of differentiating biological phenotypes as well as for validating those findings in the context of three novel cohorts of patients with Hepatitis C.ConclusionsMass-spectrometry proteomics is quickly becoming a powerful tool for studying biological and translational questions. Making use of all of the information contained in a particular set of data will be critical to the success of those endeavors. Our proposed model represents an advance in the ability of statistical models of proteomic data to identify and utilize correlation between features. This allows validation of predictors without translation to targeted assays in addition to informing the choice of targets when it is appropriate to generate those assays.

Project description:BackgroundSWATH-MS has emerged as the strategy of choice for biomarker discovery due to the proteome coverage achieved in acquisition and provision to re-interrogate the data. However, in quantitative analysis using SWATH, each sample from the comparison group is run individually in mass spectrometer and the resulting inter-run variation may influence relative quantification and identification of biomarkers. Normalization of data to diminish this variation thereby becomes an essential step in SWATH data processing. In most reported studies, data normalization methods used are those provided in instrument-based data analysis software or those used for microarray data. This study, for the first time provides an experimental evidence for selection of normalization method optimal for biomarker identification.MethodsThe efficiency of 12 normalization methods to normalize SWATH-MS data was evaluated based on statistical criteria in 'Normalyzer'-a tool which provides comparative evaluation of normalization by different methods. Further, the suitability of normalized data for biomarker discovery was assessed by evaluating the clustering efficiency of differentiators, identified from the normalized data based on p-value, fold change and both, by hierarchical clustering in Genesis software v.1.8.1.ResultsConventional statistical criteria identified VSN-G as the optimal method for normalization of SWATH data. However, differentiators identified from VSN-G normalized data failed to segregate test and control groups. We thus assessed data normalized by eleven other methods for their ability to yield differentiators which segregate the study groups. Datasets in our study demonstrated that differentiators identified based on p-value from data normalized with Loess-R stratified the study groups optimally.ConclusionThis is the first report of experimentally tested strategy for SWATH-MS data processing with an emphasis on identification of clinically relevant biomarkers. Normalization of SWATH-MS data by Loess-R method and identification of differentiators based on p-value were found to be optimal for biomarker discovery in this study. The study also demonstrates the need to base the choice of normalization method on the application of the data.

Dataset Information

Improved normalization of systematic biases affecting ion current measurements in label-free proteomics data.

Publications

Improved normalization of systematic biases affecting ion current measurements in label-free proteomics data.

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