posted on 2020-03-05, 13:09authored byMark V. Ivanov, Julia A. Bubis, Vladimir Gorshkov, Irina A. Tarasova, Lev I. Levitsky, Anna A. Lobas, Elizaveta M. Solovyeva, Marina L. Pridatchenko, Frank Kjeldsen, Mikhail V. Gorshkov
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.