Data Processing Has Major Impact on the Outcome of Quantitative Label-Free LC-MS Analysis

Abstract

High-throughput multiplexed protein quantification using mass spectrometry is steadily increasing in popularity, with the two major techniques being data-dependent acquisition (DDA) and targeted acquisition using selected reaction monitoring (SRM). However, both techniques involve extensive data processing, which can be performed by a multitude of different software solutions. Analysis of quantitative LC-MS/MS data is mainly performed in three major steps: processing of raw data, normalization, and statistical analysis. To evaluate the impact of data processing steps, we developed two new benchmark data sets, one each for DDA and SRM, with samples consisting of a long-range dilution series of synthetic peptides spiked in a total cell protein digest. The generated data were processed by eight different software workflows and three postprocessing steps. The results show that the choice of the raw data processing software and the postprocessing steps play an important role in the final outcome. Also, the linear dynamic range of the DDA data could be extended by an order of magnitude through feature alignment and a charge state merging algorithm proposed here. Furthermore, the benchmark data sets are made publicly available for further benchmarking and software developments.

Targeted Proteomics Approach for Precision Plant Breeding

“…For each run, 2 µL of sample was injected using an LC mobile phase gradient as described earlier. 27 …”

Is label‐free LC‐MS/MS ready for biomarker discovery?

“…Samples “old” and “new” were analyzed 22 months apart. “Old” samples are from the dilution series, data were published earlier . The data for “new” samples were generated from the same sample aliquots that were used for the “old” samples.…”

“…The data for “new” samples were generated from the same sample aliquots that were used for the “old” samples. dil8 and dil9 represent the samples in the dilution series as described in . Injection volumes for samples are as indicated.…”

“…Proper data processing is another key to successful protein quantification using LC-MS/MS [20]. There are multiple platforms for processing of label-free LC-MS/MS data, reviewed in recent publications [21,22].…”

“…As biomarker discovery studies in some cases inevitably will involve data acquisition over long time periods, it may be impossible to keep instrument performance stable. To illustrate this, we reanalyzed two samples consisting of spikein peptides in cell-lysate background from a recent study [20]. New data acquisition was performed 22 months after the original analysis, and the LC-MS/MS system had undergone major maintenance in between these runs.…”