title: Complete chemical modification of amine and acid functional groups of peptides and small proteins.
abstract: The chemical modification of protein thiols by reduction and alkylation is common in the preparation of proteomic samples for analysis by mass spectrometry (MS). Modification at other functional groups has received less attention in MS-based proteomics. Amine modification (Lys, N-termini) by reductive dimethylation or by acylation (e.g., iTRAQ labeling) has recently gained some popularity in peptide-based approaches (bottom-up MS). Modification at acidic groups (Asp, Glu, C-termini) has been explored very minimally. Here, we describe a sequential labeling strategy that enables complete modification of thiols, amines, and acids on peptides or small intact proteins. This method includes (1) the reduction and alkylation of thiols, (2) the reductive dimethylation of amines, and (3) the amidation of acids with any of several amines. This chemical modification scheme offers several options both for the incorporation of stable isotopes for relative quantification and for improving peptides or proteins as MS analytes.
authors: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA. ckrusema@stanford.edu
authors: Casey J
authors: CJ
authors: Krusemark
authors: Brian L
authors: BL
authors: Frey
authors: Lloyd M
authors: LM
authors: Smith
authors: Peter J
authors: PJ
authors: Belshaw
references: J Biol Chem. 1967 May 25;242(10):2447-53
references: 6026234
references: Anal Chem. 2004 Oct 1;76(19):5894-902
references: 15456312
references: Anal Biochem. 1999 Jul 1;271(2):197-9
references: 10419639
references: Science. 1992 Sep 25;257(5078):1885-94
references: 1411504
references: Nat Biotechnol. 2002 Mar;20(3):301-5
references: 11875433
references: J Am Chem Soc. 2003 Oct 15;125(41):12392-3
references: 14531666
references: Anal Biochem. 1995 Jan 1;224(1):1-16
references: 7710054
references: J Am Soc Mass Spectrom. 2009 Dec;20(12):2183-91
references: 19747844
references: Anal Chem. 2008 Nov 1;80(21):8324-8
references: 18821779
references: J Am Chem Soc. 2004 Aug 4;126(30):9152-3
references: 15281783
references: Proteomics. 2005 Jan;5(1):4-15
references: 15602776
references: Anal Chem. 2005 Dec 1;77(23):7783-95
references: 16316189
references: Anal Chem. 2007 Jan 15;79(2):673-81
references: 17222036
references: Anal Chem. 2008 Feb 1;80(3):713-20
references: 18184016
references: Nat Protoc. 2009;4(4):484-94
references: 19300442
references: Anal Chem. 2004 May 15;76(10):2748-55
references: 15144184
references: J Proteome Res. 2008 Sep;7(9):4178-82
references: 18636758
references: Mol Cell Proteomics. 2004 Dec;3(12):1154-69
references: 15385600
references: J Am Soc Mass Spectrom. 2009 Sep;20(9):1617-25
references: 19481956
references: Int J Mass Spectrom. 2008 Oct 1;276(2-3):136-143
references: 19802328
references: Anal Chem. 2006 Feb 1;78(3):686-94
references: 16448040
references: J Chromatogr. 1992 Sep 11;608(1-2):239-42
references: 1430027
references: J Am Soc Mass Spectrom. 2000 Jan;11(1):78-82
references: 10631667
references: Acta Chem Scand. 1999 Dec;53(12):1122-6
references: 10629937
references: Rapid Commun Mass Spectrom. 2001;15(14):1214-21
references: 11445905
references: Biochem J. 1982 Apr 1;203(1):331-4
references: 7103947
references: J Proteome Res. 2007 Dec;6(12):4770-6
references: 18001078
references: Science. 2006 Oct 6;314(5796):65-6
references: 17023639