Deep and Highly Sensitive Proteome Coverage by LC-MS/MS Without Prefractionation

Abstract

In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795–806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.

Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

“…This, however, requires that the phosphopeptide enrichment step be of exquisite selectivity, have sufficient capacity, and allow complete elution (17,18). Phosphopeptides purified in this way can be directly analyzed via LC-MS/MS (33,34), typically using shallow reversed phase LC gradients (35). However, it turns out that current mass spectrometers still lack the scan speed and dynamic range required to reach complete (phospho)peptide sampling in direct LC-MS/MS measurements (36,37).…”

A Fast Workflow for Identification and Quantification of Proteomes

“…S2A and S2B). The number of proteins identified in this workflow with 30-min gradient is no fewer than those from longer gradient (7,8). We detected 73,680 unique peptides on QE from the 12-h run, which resulted in 21.26% median sequence coverage of identified proteins.…”

“…Current pro- teomics strategy tends to use prefractionation and to employ long gradient in the second dimension HPLC/MS/MS to overcome the problem of sample complexity (8), this is achieved at the expense of MS running time, such that a typical experiment for proteome coverage takes days and weeks to complete. Such long time of MS running is cost prohibitive for many scientists and is challenging to maintain the instrument at constant conditions during different runs so that the results can be compared.…”

Post-translational Modifications and Mass Spectrometry Detection

“…Several workflows have been developed, following the pioneering work of Yates and co-workers in an approach named multidimensional protein identification technology (MudPIT) (79). Recently, the shotgun approach was used in the analysis of yeast proteomes, allowing the identification of 2990 yeast proteins corresponding to 35,155 sequence unique peptides, and average sequence coverage rate of 18% (80). Although this workflow allows high throughput protein identification, the assignment of PTM sites is compromised due to the MS dynamic range and the typical low sequence coverages that are obtained.…”