A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.
Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.
Liu
Peiran
P
Department of Biologics Product and Process Development, Bristol-Myers Squibb Technical Operations, Princeton, New Jersey 08543, USA.
O'Mara
Brian W
BW
Warrack
Bethanne M
BM
Wu
Wei
W
Huang
Yunping
Y
Zhang
Yihong
Y
Zhao
Rulin
R
Lin
Mei
M
Ackerman
Michael S
MS
Hocknell
Peter K
PK
Chen
Guodong
G
Tao
Li
L
Rieble
Siegfried
S
Wang
Jack
J
Wang-Iverson
David B
DB
Tymiak
Adrienne A
AA
Grace
Michael J
MJ
Russell
Reb J
RJ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D003545:Cysteine; D010446:Peptide Fragments; D010720:Phosphines; D011506:Proteins; C080938:tris(2-carboxyethyl)phosphine