Unambiguous determination of isobaric histone modifications by reversed-phase retention time and high-mass accuracy.
Methylation and acetylation of lysines are crucial posttranslational modifications that regulate gene transcription and have been shown to be misregulated in many forms of cancers. Western blot, immunoprecipitation, and immunofluorescence are commonly used to characterize histone acetylation and methylation. However, these approaches are limited by the availability, site specificity, and cross-reactivity of antibodies. Mass spectrometry is emerging as an additional powerful tool for histone characterization. The isobaric nature of trimethylation and acetylation (42.0470 and 42.0106 Da, respectively) confounds histone characterization by means other than high-resolution/high-mass accuracy mass spectrometry. In this study, we adapted methodology that exploits difference in the relative retention time of acetylated and methylated peptides to unequivocally distinguish between these two modifications even with low-mass accuracy mass spectrometers. The approach was tested on tryptic digest of Saccharomyces cerevisiae histones. We found that acetylation resulted in increased retention in reversed-phase chromatography, whereas methylation, including trimethylation, showed little change in retention. For example, the acetylated forms of peptide (27)KSAPSTGGVKKPHR(40) eluted at 15.63 min, whereas the methylated forms eluted at 13.89 min. In addition, the effect of acetylation was cumulative as observed in the case of peptide (9)KSTGGKAPR(17), whose unmodified, monoacetylated, and diacetylated isoforms eluted at 7.43, 10.47, and 16.49 min, respectively. The modification patterns of the peptides in question were subsequently verified by high-mass accuracy tandem mass spectrometry.
Yang
Lanhao
L
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
Tu
Shengjiang
S
Ren
Chen
C
Bulloch
Esther M M
EM
Liao
Chung-Lin
CL
Tsai
Ming-Daw
MD
Freitas
Michael A
MA
D000107:Acetylation; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors
D006657:Histones; D010455:Peptides
J Chromatogr. 1993 Aug 27;646(1):17-30
8408425
Mol Cell Proteomics. 2002 Jul;1(7):500-8
12239278
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14440-5
8962070
Curr Opin Genet Dev. 1999 Apr;9(2):171-4
10322142
Curr Opin Genet Dev. 2005 Apr;15(2):163-76
15797199
J Biol Chem. 2007 May 11;282(19):14262-71
17369256
J Proteome Res. 2007 Jan;6(1):81-8
17203951
J Biol Chem. 2005 Mar 18;280(11):10264-76
15632193
PLoS One. 2007 Nov 21;2(11):e1210
18030344
Chromosoma. 2003 Aug;112(2):77-86
12937907
Cell. 2005 Nov 18;123(4):593-605
16286008
Mol Cell Proteomics. 2007 Apr;6(4):677-88
17205979
Cell. 2005 Aug 26;122(4):517-27
16122420
J Mass Spectrom. 1998 Jan;33(1):1-19
9449829
Nat Methods. 2004 Nov;1(2):119-26
15782174
BMC Bioinformatics. 2007 Apr 20;8:133
17448237
Cell. 2005 May 6;121(3):375-85
15882620
Proteomics. 2004 Jan;4(1):1-10
14730666
Methods. 2003 Sep;31(1):3-11
12893168
J Chromatogr A. 2002 May 31;957(2):187-99
12113342
J Chromatogr. 1988 Dec 23;458:193-205
3235635
Microbiol Mol Biol Rev. 2000 Jun;64(2):435-59
10839822
Proc Natl Acad Sci U S A. 1980 Mar;77(3):1632-6
6929513
Proteomics Clin Appl. 2007 Jan;1(1):130-42
21136616
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 May 1;850(1-2):440-54
17254850
J Cell Biochem. 2005 Apr 15;94(6):1088-102
15723344
J Cell Sci. 2003 Jun 1;116(Pt 11):2117-24
12730288
J Am Soc Mass Spectrom. 2005 Oct;16(10):1641-53
16099169
Anal Chem. 2006 Nov 15;78(22):7785-95
17105172
Cell. 2004 Jun 11;117(6):721-33
15186774
Mol Cell Proteomics. 2004 Sep;3(9):872-86
15199121
Anal Biochem. 2003 May 1;316(1):23-33
12694723
Nat Protoc. 2007;2(4):933-8
17446892
J Am Soc Mass Spectrom. 2003 Sep;14(9):980-91
12954166
Anal Chem. 2002 Nov 15;74(22):5826-30
12463368
Expert Rev Proteomics. 2007 Apr;4(2):211-25
17425457
Methods Enzymol. 2004;377:130-53
14979022
J Proteome Res. 2004 May-Jun;3(3):621-6
15253445
Proteomics. 2004 May;4(5):1382-96
15188406
Anal Chem. 2003 Mar 1;75(5):1039-48
12641221
Mol Cell. 2000 Jun;5(6):905-15
10911985
Eur J Biochem. 1990 Nov 13;193(3):701-13
2249688
Nat Rev Mol Cell Biol. 2006 Sep;7(9):657-66
16912715
J Biol Chem. 2002 Aug 16;277(33):29496-502
12039950
Mol Cell Biol. 2006 Nov;26(21):8122-35
16923962
Mol Cell Proteomics. 2006 Jan;5(1):172-81
16204703
Mol Cell. 2000 Jun;5(6):917-26
10911986
Nat Rev Mol Cell Biol. 2005 Nov;6(11):838-49
16261189
Mol Cell. 2003 Dec;12(6):1577-89
14690609
J Cell Biochem. 2004 Jul 1;92(4):691-700
15211567