여름정기학술대회
2022여름초록
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Proteomics aims to deep proteome profiling in tissues, cells,
or body fluids to understand the biological processes and signaling pathways.
The bottom-up proteomics analyzes complex peptides mixture after enzymatic
digestion of protein. These peptides in single sample may contain millions of peptides
with dynamic range of more than 10 orders. In order to overcome high complexity
and wide dynamic range of sample, many proteomics studies employ a
fractionation method, such as non-contiguous fractionating and concatenating
(NCFC) fractionation utilizing high-pH RPLC, before each fraction is subjected
to LC-MS/MS experiments. These NCFC fractionation method was usually performed
offline with many manual steps, which result in sample loss and less
reproducibility.
In this study, we present a dual online
reverse-phase/reverse-phase liquid chromatography system that employs an online
non-contiguous fractionating and concatenating device (NCFC fractionator). The DO-2D-NCFC-RP/RPLC
shows significant improvements in separation spaces by improved orthogonality
of two RPLCs (mid-pH and low-pH RPLC) and “hidden” isocratic elution implemented for the last
dimension RPLC by the mutually independent operation of the dual online RPLC.
The DO-2D-NCFC-RP/RPLC system produces up to 24 NCFC online fractions and each
fraction was subsequently analyzed by the dual online RPLC-MS/MS experiments. The
DO-2D-NCFC-RP/RP-MS/MS experiments were fully automated by a method script that
time-controls the valve positions. As a result, this
system provides minimal sample loss and high reproducibility. From 25 mg of HeLa
peptides, this novel system identified more than 10,000 proteins in one day of MS data acquisition
time. Also, by combining
DO-2D-NCFC-RP/RPLC with online high-field asymmetric waveform ion mobility
spectrometry (FAIMS), the resultant online 3D separation resulted in
identification of more than 100,000 phosphopeptides (>60,000 phosphosites) from
3 mg of HeLa peptides, the largest phosphoproteome reported to data from HeLa
cells.
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