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Advantages of proteomics using meter‐long monolithic columns with small inner diameter
Author(s) -
Ohtani Yuta,
Aburaya Shunsuke,
Minakuchi Hiroyoshi,
Miura Natsuko,
Aoki Wataru,
Ueda Mitsuyoshi
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.475.8
Subject(s) - chromatography , analyte , analytical chemistry (journal) , mass spectrometry , chemistry , elution , sensitivity (control systems) , monolithic hplc column , materials science , particle size , high performance liquid chromatography , electronic engineering , engineering
Background Targeted proteomics by selected reaction monitoring (SRM) with high signal‐noise ratio enables to quantify hundreds of proteins with high sensitivity. However, since the lowest number of proteins is less than 10 copies in a single cell, further improvement from the aspects of sensitivity is necessary to detect these ones. Methods Unlike conventional particle‐packed columns used in liquid chromatography (LC), monolithic columns are characterized by an orderly 3D‐network structure [1]. With the monolithic columns, high‐performance separation with a thin skeleton and back pressure can be achieved. Further improvement in detection‐sensitivity is achieved by reducing column inner diameter (I.D.), because small elution volume concentrates analytes and efficient desolvation increases ionization efficiency. Hence, we developed meter‐long monolithic columns with small I.D. to SRM‐based proteomics and aimed to improve detection‐sensitivity of mass spectrometry (MS) to increase peak intensity in peptide levels. BSA tryptic digests were directly injected into monolithic columns with different length (500 mm or 1000 mm) and I.D. (100 μm or 75 μm), and peak capacity and peak area were evaluated. Results & Discussion Separation efficiency increased 1.9 times by lengthening a monolithic column from 500 mm to 1000 mm. Detection‐sensitivity increased 2.3 times in peptide level by reducing column I.D. from 100 μm to 75 μm. Furthermore, even when column I.D. was reduced, there were no differences in the coefficient of variation values of the peak area. These results showed reduction of I.D. of meter‐long monolithic columns increased detection‐sensitivity in SRM‐based proteomics [2]. Support or Funding Information This work was supported by JST CREST (Grant Number JPMJCR16G2), Japan. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .