Proteome sequencing goes deep

Abstract

Advances in mass spectrometry (MS) have transformed the scope and impact of protein characterization efforts. Identifying hundreds of proteins from rather simple biological matrices, such as yeast, was a daunting task just a few decades ago. Now, expression of more than half of the estimated ∼20,000 human protein coding genes can be confirmed in record time and from minute sample quantities. Access to proteomic information at such unprecedented depths has been fueled by strides in every stage of the shotgun proteomics workflow-from sample processing to data analysis-and promises to revolutionize our understanding of the causes and consequences of proteome variation.

Figure 1

Workflow for “shotgun” or “bottom-up” proteomics. (a) Preparing proteomic samples for LC-MS/MS analysis requires protein extraction, proteolysis, and, optionally, peptide-level fractionation. (b) Online LC separation of complex peptide mixtures introduces analytes into the mass spectrometer for precursor and fragment ion mass analysis. (c) Tandem mass spectra are matched to theoretical spectra generated in silico to garner peptide sequences that are used for protein inference.

Figure 2

Chromosomal coverage of the human proteome (reproduced with permission from ref. [17]). In one of two recent large-scale investigations of the human proteome, Wilhelm and coworkers identified 18,097 proteins, covering over 90% of all but three chromosomes (11, 21, and Y). The density of proteins covered in any particular chromosomal region is indicated by the blue bars.