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High‐sensitivity TFA‐free LC‐MS for profiling histones
Author(s) -
You Jia,
Wang Liwen,
Saji Motoyasu,
Olesik Susan V.,
Ringel Matthew D.,
Lucas David M.,
Byrd John C.,
Freitas Michael A.
Publication year - 2011
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201000445
Subject(s) - chemistry , histone , adduct , chromatography , formic acid , label free quantification , biochemistry , quantitative proteomics , proteomics , dna , organic chemistry , gene
The analysis of proteins by RPLC commonly involves the use of TFA as an ion‐pairing agent, even though it forms adducts and suppresses sensitivity. The presence of adducts can complicate protein molecular weight assignment especially when protein isoforms coelute as in the case of histones. To mitigate the complicating effects of TFA adducts in protein LC‐MS, we have optimized TFA‐free methods for protein separation. Protein standards and histones were used to evaluate TFA‐free separations using capillary (0.3 mm id) and nanoscale (0.1 mm id) C 8 columns with the ion‐pairing agents, formic acid or acetic acid. The optimized method was then used to examine the applicability of the approach for histone characterization in human cancer cell lines and primary tumor cells from chronic lymphocytic leukemia patients.