DirectMS1: MS/MS-Free Identification of 1000 Proteins of Cellular Proteomes in 5 Minutes

Proteome
characterization relies heavily on tandem mass spectrometry
(MS/MS) and is thus associated with instrumentation complexity, lengthy
analysis time, and limited duty cycle. It was always tempting to implement
approaches that do not require MS/MS, yet they were constantly failing
to achieve a meaningful depth of quantitative proteome coverage within
short experimental times, which is particularly important for clinical
or biomarker-discovery applications. Here, we report on the first
successful attempt to develop a truly MS/MS-free method, DirectMS1,
for bottom-up proteomics. The method is compared with the standard
MS/MS-based data-dependent acquisition approach for proteome-wide
analysis using 5 min LC gradients. Specifically, we demonstrate identification
of 1 000 protein groups for a standard HeLa cell line digest.
The amount of loaded sample was varied in a range from 1 to 500 ng,
and the method demonstrated 10-fold higher sensitivity. Combined with
the recently introduced Diffacto approach for relative
protein quantification, DirectMS1 outperforms most popular MS/MS-based
label-free quantitation approaches because of significantly higher
protein sequence coverage.