Nanoscale LC is most compatible with high-resolution MS with useful, extremely complex chromatograms with as little as 0.1 ng sample derived from less than 10 mammalian cells.
As presented in a poster by Yufeng Shen et al.—“Cutting-Edge Nano LC Column Technology and its Analytical Capabilities in Advanced Tandem Mass Spectrometry Proteomics” (CoAnn Technologies, LLC, Richland, WA); Poster CP051 at ASMS 2018—the chromatograms showed over 6500 proteins and more than 55,000 peptides in a single run of 500 minutes. The column was 60 cm × 75 µm i.d. filled with a 1.7-µm C18 column running at 8000 psi.
Applications reported included top-down MS/MS of more than 10,000 proteoforms from over 1500 proteins in a single run. The number of identified proteoforms can be increased by pre-HPLC fractionation. This can be useful in discriminating between the biological activity of different proteoforms.
Phosphorylation is a common post-translational modification of proteins. The poster showed chromatograms from 20- and 60-cm-long columns. The latter provided a complex chromatogram to which the MS/MS assigned 20,288 peptides (FDR <1%) in a single 500-minute run. The MS was an Orbitrap Elite (hybrid ion trap-Orbitrap mass spectrometer) from Thermo Fisher Scientific.
This work is typical of the high-resolution HPLC/MS for proteomics developed in the laboratory of Richard Smith at Pacific Northwest National Laboratory (Richland, WA). Dr. Smith was listed as a coauthor.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]
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