Pitfalls in Proteomics Avoiding Problems That Can Occur Before Data Acquisition Begins

Abstract

Nano-LC-MS has become the gold-standard methodology used for proteomic analysis (defined here as the identification and quantification of protein abundance in biological samples) due its ability to provide sensitive detection for untargeted peptides, as well as identification of unknown peptides through MS/MS fragmentation and comparison of the experimental spectra to theoretical spectra prepared in-silico. Subsequently, nano-LC-MS-based proteomics tools have become invaluable in diverse disciplines ranging from immunology to microbiology, and food chemistry. However, analysis of biomolecules is generally susceptible to a number of unique and perilous sample preparation itfalls that can severely degrade the quality of data acquired, even when the most sophisticated LC-MS systems are used. This is due in part to the unique chemistry and size of large biological macromolecules such as proteins, peptides and oligonucleotides to name a few. Herein, we provide our views on some of the most common pitfalls in sample and instrument preparation relevant to proteomic analyses by nano-LC-MS. We hope that increasing awareness of these potential problems can help users mitigate them and produce higher quality data in general. While we focus here on nano-scale separations wherein peak volumes are exceptionally low and susceptible to contamination, these tips can be applied to any form of LC-MS biomolecule analysis.