Full Dynamic Range Proteome Analysis of S. cerevisiae by Targeted Proteomics

Abstract

Summary The rise of systems biology implied a growing demand for highly sensitive techniques for the fast and consistent detection and quantification of target sets of proteins across multiple samples. This is only partly achieved by classical mass spectrometry or affinity-based methods. We applied a targeted proteomics approach based on selected reaction monitoring (SRM) to detect and quantify proteins expressed to a concentration below 50 copies/cell in total S. cerevisiae digests. The detection range can be extended to single-digit copies/cell and to proteins which were undetected by classical methods. We illustrate the power of the technique by the consistent and fast measurement of a network of proteins spanning the entire abundance range over a growth time-course of S. cerevisiae transiting through a series of metabolic phases. We therefore demonstrate the potential of SRM-based proteomics to provide assays for the measurement of any set of proteins of interest in yeast at high-throughput and quantitative accuracy.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…After using these heavy reference peptides for optimizing collision energies for all selected SRM transitions, the linear dynamic range, reproducibility, and quantification limits of each SRM assay were thoroughly assessed through analyses of dilution series with whole human cell lysates (Bauer et al , 2014). Although wide dynamic ranges were observed for all assays (> 4 orders of magnitude), extract fractionation by Off‐Gel electrophoresis was required to further improve the sensitivity of SRM assays (Picotti et al , 2009) and allow confident detection and quantification of the least abundant target proteins. By comparing transition intensities of “heavy” reference peptides (spiked in known amounts into tryptic digests prior to any fractionation) and their corresponding “light” counterparts derived from endogenous proteins (Appendix Fig S3), the abundance of endogenous proteins in whole‐cell extracts could be accurately determined (Bauer et al , 2014).…”

“…In particular, selected reaction monitoring (SRM) allows the detection of specific proteins in complex mixtures with high sensitivity and over a broad dynamic range (Picotti et al , 2009; Bauer et al , 2014). In parallel, EGFP‐tagging of endogenous proteins through genetic engineering offers opportunities for quantifying fluorescence signals at specific subcellular locations (e.g.…”

Applications of targeted proteomics in systems biology and translational medicine

“…One of the earliest perturbed protein network study was carried out by Picotti and colleagues quantifying proteins of the Krebs Cycle under diauxic shift in Saccharomyces cerevisiae 16. The methodology quickly spread to medium sized target lists and has been used in a variety of basic biology applications, including pharmacology and developmental biology.…”

“…Following the successful quantification of single proteins or small protein lists, assays for targeted proteomics were established for entire model organisms 16, 46, 47. Applications using medium to large‐sized target lists as is the case for the study of biological networks have pushed technology development.…”

“…Measuring proteins poses technical challenges, particularly in higher eukaryotes which are made‐up of trillions of cells that are categorized, somewhat arbitrarily, into more than 400 different cell types 13. The two foremost challenges are the sheer number of proteins present in a cell and their vast dynamic range of expression which spans four to five orders of magnitude in prokaryotes, —six to seven orders of magnitude in eukaryotic cells/tissues and 12 orders of magnitude in body fluids 14, 15, 16. Moreover, proteins are subject to more than 200 types of PTMs, which further magnify the number of distinct protein entities in a sample and sample handling chemistries 17, 18.…”