Now, More Than Ever, Proteomics Needs Better Chromatography

Abstract

From plant research to biomedicine, proteome analysis plays a critical role in many areas of biological inquiry. Steady improvement in mass spectrometer (MS) technology has transformed the speed and depth of proteome analysis. Proteomes of simple organisms can now be sequenced to near completion in just over an hour. Comparable coverage of mammalian proteomes, however, still requires hours or even days of analysis. Here we ask why current technology fails to achieve comprehensive and rapid analysis of the more complex mammalian proteomes. We propose that further advancements in MS technology alone are unlikely to solve this problem and suggest that concomitant improvements in peptide separation technology will be critical.

An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes

“…This is not extraordinarily impressive, but when 46 fractions are analyzed together this translates into a very high peak capacity estimation of 13,300. The lack of full orthogonality between separations in the two LC dimensions lowers the actual peak capacity value (Figure 2D), but it is still one order of magnitude higher than the best one-dimensional LC separations that may approach a peak capacity of 1,000 on long columns/gradients (Shishkova et al., 2016). The offline HpH fractionation was performed with high resolution, as 82% of peptide spectrum matches and 75% of peptide sequences were unique to a single HpH fraction, and a further 17% of peptide sequences were only found in two fractions as indicated by the gray scale in the graphical representation (Figure 2E).…”

A Novel Differential Ion Mobility Device Expands the Depth of Proteome Coverage and the Sensitivity of Multiplex Proteomic Measurements

“…Improvements in quadrupole performance through higher resolution and scanning rate have enabled more efficient isolation and transmission of target ions with narrow isolation windows in turn reducing chimeric tandem mass spectra (12). Newer generation of MS instruments that combine high resolution of survey scan with fast scanning rate of MS/MS acquisition are progressively bridging the gap that exists between detectable and identifiable peptide ions (13,14). The higher resolution of MS instruments also highlights the presence of convoluted isobaric ions with unassigned charge states that prevent their selection for MS/MS acquisition.…”

Comprehensive Single-Shot Proteomics with FAIMS on a Hybrid Orbitrap Mass Spectrometer

“…We believe that the central limitation in these analyses is separation peak capacity. 4 This conclusion is evinced by the common use of two-dimensional liquid chromatography (2DLC) for complex protein mixtures. In this practice each fraction is analyzed separately by nano liquid chromatography–tandem mass spectrometry (nanoLC–MS/MS), resulting in increased total peak capacity and increased proteomic depth.…”