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Phenyl‐bonded monolithic silica capillary column liquid chromatographic separation and detection of fluorogenic derivatized intact proteins
Author(s) -
Kobayashi Hiroshi,
Wada Hiroo,
Imai Kazuhiro
Publication year - 2021
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.5078
Subject(s) - chemistry , chromatography , proteome , reagent , mass spectrometry , capillary action , proteomics , biochemistry , organic chemistry , materials science , composite material , gene
Abstract Prior to the identification of proteins for proteomics analysis in human cells, separation of fluorogenic derivatized proteins with a fluorogenic reagent, 7‐chloro‐ N ‐[2‐(dimethylamino)ethyl]‐2,1,3‐benzoxadiazole‐4‐sulfonamide, has typically been performed by using a conventional reversed‐phase HPLC column. However, the number of proteins in human cells (HepaRG) that are separated by this conventional approach is limited to approximately 500. In this study, a nanoflow liquid chromatography system with an evaluated phenyl‐bonded monolithic silica capillary column (0.1 mm i.d., 700 mm length) was used to increase the number of separated fluorogenic derivatized proteins. This system was used to separate derivatized human cell proteins (K562) and yeast ( Saccharomyces cerevisiae ) proteins as model cell proteomes. More than 1,300 protein peaks were separated/detected from both cell proteomes. We present a straightforward comparison of multiple separation profiles using a novel chromatogram display approach, termed the “spiderweb” chromatogram. In addition, to validate that the detected peaks are derived from proteins, a mass spectrometer was connected to the capillary column and deconvolution of the obtained mass spectra was performed. Furthermore, different molecular weight distribution profiles of the expressed proteins were observed between the two cell proteomes.