Probing Lysine Acetylation in Proteins

The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF). The acetylation of proteins at specific lysine residues by acetyltransferase enzymes has emerged as a posttranslational modification of high biological impact. Although lysine acetylation in histone proteins is an integral part of the histone code the acetylation of a multitude of non-histone proteins was recently recognized as a regulatory signal in many cellular processes. New substrates of acetyltransferase enzymes are continuously identified, and the analysis of acetylation sites in proteins is increasingly performed by mass spectrometry. However, the characterization of lysine acetylation in proteins using mass spectrometric techniques has some limitations and pitfalls. The non-enzymatic cysteine acetylation especially can result in false-positive identification of acetylated proteins. Here we demonstrate the application of various mass spectrometric techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry for the analysis of protein acetylation. We describe diverse combinations of biochemical methods useful to map the acetylation sites in proteins and discuss their advantages and limitations. As an example, we present a detailed analysis of the acetylation of the HIV-1 transactivator of transcription (Tat) protein, which is known to be acetylated in vivo by the acetyltransferases p300 and p300/CBP-associated factor (PCAF). The acetylation of proteins by acetyltransferases is increasingly considered a biologically relevant regulatory modification like phosphorylation (1Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation?.EMBO J. 2000; 19: 1176-1179Google Scholar). Acetyltransferases transfer acetyl groups from acetyl-coenzyme A (AcCoA) 1The abbreviations used are: AcCoA, acetyl-coenzyme A; aa, amino acids; ARM, arginine-rich motif; CRR, cysteine-rich region; HIV, human immunodeficiency virus; PCAF, p300/CBP-associated factor; CBP, cAMP-response element-binding protein (CREB)-binding protein; PTH, phenylthiohydantoin; Tat, transactivator of transcription; Fmoc, N-(9-fluorenyl)methoxycarbonyl. 1The abbreviations used are: AcCoA, acetyl-coenzyme A; aa, amino acids; ARM, arginine-rich motif; CRR, cysteine-rich region; HIV, human immunodeficiency virus; PCAF, p300/CBP-associated factor; CBP, cAMP-response element-binding protein (CREB)-binding protein; PTH, phenylthiohydantoin; Tat, transactivator of transcription; Fmoc, N-(9-fluorenyl)methoxycarbonyl.either to the α-amino group of the amino-terminal residue (N-acetyltransferases) or to the ε-amino group of specific lysine residues (histone/factor acetyltransferases) of substrate proteins. The reverse reaction is catalyzed by deacetylases that remove acetyl groups from specific acetyllysine residues in their substrates. The reversible lysine acetylation of histones and non-histone proteins plays a vital role in the regulation of many cellular processes including chromatin dynamics and transcription (2Kuo M.H. Allis C.D. Roles of histone acetyltransferases and deacetylases in gene regulation.Bioessays. 1998; 20: 615-626Google Scholar, 3Kouzarides T. Histone acetylases and deacetylases in cell proliferation.Curr. Opin. Genet. Dev. 1999; 9: 40-48Google Scholar, 4Knoepfler P.S. Eisenman R.N. Sin meets NuRD and other tails of repression.Cell. 1999; 99: 447-450Google Scholar, 5Wolffe A.P. Guschin D. Chromatin structural features and targets that regulate transcription.J. Struct. Biol. 2000; 129: 102-122Google Scholar), gene silencing (6Bestor T.H. Gene silencing. Methylation meets acetylation.Nature. 1998; 393: 311-312Google Scholar, 7Razin A. 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Scholar). lysine residues as during the The and proteins and from the with by and by or of a 1 Tat with an the mass an of in the with a with a and a The at the was to at the to and at the to by to laser The using the of and and and the of and the proteins in to a of 5 and the mass A of was by a and with an of used for the of the with the of the mass the a of was to the and the was to V. in the an of and a of of acetylated in vitro with of in the or of of p300 or PCAF acetyltransferase acetyltransferase as A. V. A. P. U. E. M. between Tat and PCAF is the binding of acetylated Tat to the PCAF J. Scholar, K. A. C. V. P. M. E. E. M. Acetylation of Tat a in Cell. 2003; 12: Scholar). was performed at for in a of of reaction 1 and The reaction was by the in vitro acetylated and using or to the with was or the mass an of in the with a II with a and a The at the was to at the to at the to and at the to by to laser The by a using of and the of the in vitro acetylated and using or to the The was in and the mass A of was as to at the and to at the of was performed in the a of and an of analysis was performed in the using the an mass The was by the was performed by using the with an of or for and the using the of the analysis of the Tat was performed by using a to a mass as A. S. A. Allen M. J. mass spectrometry for Chem. 2004; Scholar). vitro acetylated Tat was in at for to remove from the acetylation the was in of and at for using of The reaction was by in vitro acetylation the Tat was and a using the to the was performed a protein using by the and the of the was by using the can be used for the of proteins that are acetylated at lysine residues of the known acetylated proteins by or is after in vivo or after in vitro acetylation of or proteins using or after of the acetylated proteins from and other that is by using can be performed with in vivo and in vitro acetylated proteins using is with to methods such as or a of for the analysis of protein acetylation is However, and for acetylation the of acetylation sites in a mass spectrometric of the of in vivo or in vitro acetylated proteins their characterization the of acetyl groups to the the signal of an acetylated protein is by acetyl group as with the signal of the protein the of acetylation sites in a protein can be using and to be techniques for the of the of proteins. the analysis of HIV-1 Tat protein by and Tat protein, Tat acetylated at and a of analyzed by The signal of Tat was at which is in with the mass for The Tat was at The signal for the acetylated Tat was by as with the signal of the Tat the of a acetyl The by analysis a mass Here the acetyl mass of was after of the mass of and and for the of acetylated and of the proteins and used in of HIV-1 of HIV-1 Tat, acetylated at of HIV-1 Tat amino-terminal of HIV-1 Tat amino-terminal acetylated at of histone acetylated at of HIV-1 Tat of HIV-1 Tat of HIV-1 Tat of HIV-1 Tat of HIV-1 Tat of HIV-1 Tat in a The identification of acetylation sites in proteins can be performed using the acetylation sites a substrate is performed using proteins as and substrates of acetyltransferases and the of acetylation sites in the vitro analysis of histone acetyltransferase 2004; Scholar). in vivo acetylated proteins and in vitro acetylated or proteins be by a like or to analysis acetylation sites of a protein are for in vitro acetylation and mass spectrometric the or that are acetylated and the of acetylation sites the the of mass the of the acetylation sites in the and techniques can be or in with a of analysis such as or and acetylated residues in the analysis a result analysis using in with or is in analysis by is a biochemical to acetylated lysine residues in a that of techniques analysis can be performed using at specific amino in with or the detailed of the acetylation of the HIV-1 Tat protein be to the and of the of the HIV-1 Tat is a transactivator of transcription that amino Tat lysine residues and is known to be acetylated in vivo by the acetyltransferases p300 and PCAF M. M. J. W. S. E. Acetylation of the HIV-1 Tat protein by p300 is for transcriptional Biol. 1999; 9: Scholar, C. L. E. H. F. C. A. Y. M. C. HIV-1 Tat transcriptional activity is regulated by J. 1999; 18: Scholar, L. C. P. Wang L. P. H. Kashanchi F. Acetylation of HIV-1 by transcription of HIV-1 and binding to 2000; Scholar). the Tat that as substrate for p300 and PCAF various of of Tat The of acetyl groups in or can be by and As in the of mass for an acetylation the of acetylation sites in the 5 the mass spectrometric analysis of the the of The and acetylated that was acetylated during the of the analyzed by and and the acetylated at the mass of in with the at A and the of the mass for a acetylation the of the for for and for the of the Tat the of the and the at and the of the at and and the of the at and analysis by or the and for the characterization of acetylation sites in a induced by and at the the in a the and K. for Scholar). The of the acetylated residues can be by the acetyl mass of of the and are during the and ionization and for the modification in to and which of and analysis by matrix-assisted laser mass Chem. 68: Scholar). However, a specific acetyllysine at is in and in to the acetyllysine at Kim Kwon Yoo lysine acetylation with a using spectrometry with Chem. and can be as that an acetylated lysine residue is present in the of are other lysine residues or the amino of the are can to between acetyllysine and the the amino in the the analysis of a histone is acetylated of and in and The of the acetyl group at was the to by at the the in the at the A and The of the acetylated amino was the mass between the and the and between the and the which was for an acetylated of for an the the Tat to the Tat was to analysis the was acetylated in vitro with in the and of the acetyltransferase p300 The of the in the of mass of the signal at an signal at was after in vitro acetylation of the in the of p300 an transfer of a acetyl group to the Tat by the signal of the at was to the acetylation of the during the in vitro signal for the was at be that of the lysine residues in the Tat ARM, or was acetylated by The the Tat was acetylated in the of PCAF which of the lysine residues was acetylated by p300 and PCAF the was to However, by by a be of and amino the to a of and the acetylation in the Tat be by and analysis was analysis is analysis result in an localization of the acetylated residue the The of the is especially useful for the analysis of lysine at the of carboxyl-terminal of are to M. M. J. W. S. E. Acetylation of the HIV-1 Tat protein by p300 is for transcriptional Biol. 1999; 9: Scholar, K. K.E. L. P. Histone acetylation and identification of acetylation and sites of histone by mass spectrometry.Mol. Cell. Scholar, W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar). is for the characterization of the acetylation in the Tat analysis or was the of acetylation by p300 and PCAF an was in of Tat acetylated at of the the acetylated amino-terminal at and the carboxyl-terminal at of the acetylated at of the the amino-terminal at and the carboxyl-terminal at the of the that was by signal at was after of the with and PCAF and in to the reaction acetyltransferase The result was with the Tat that was acetylated in the of p300 The signal to the of with from of of lysine the acetyl in is the acetylated signal was by the of the of the at and at to the acetylation signal was for the at acetylation of was as the acetylation of PCAF and p300 in the Tat by to map the of lysine acetylation in a is analysis by Here the amino-terminal amino of a are with from the the and by their in J. as for characterization of 1997; Scholar, A and protein Biol. Chem. Scholar). the amino the of an lysine residue the and the a a from the modification of the amino of the and an from the modification of the ε-amino group in the lysine the of an acetylated lysine residue a the acetyl group to the lysine the modification and the of the W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar, S. M. L. silencing and protein is an histone 2000; Scholar). The of the amino the acetyllysine and a other amino an the acetylated lysine in a can be by the identification of as the acetylation of PCAF in the Tat the reaction performed in the of PCAF and a to the lysine in the amino However, after of PCAF to the in vitro acetylation a for with a of is for the of an acetylated lysine residue as was with a an acetyllysine the specific for the lysine residues was the specific for acetylated was present at a and of the protein are in J. as for characterization of 1997; was for an acetylation of by The result was after acetylation of the Tat with p300 W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar). that in the Tat is acetylated by PCAF and The for the identification of acetylation sites in can for analysis by or be or the to to their amino and and can result in for an some amino are or to the of the reaction and especially are to by and to their and the of is analysis is and and considered is to the acetylation sites in a to a amino that acetyl the of Tat to the in which and by and in vitro acetylated with in the and of the acetyltransferase The mass of the the to the signal of the at signal be at that acetylation was in the mass by from to be in the of the with the to The after acetylation of the Tat with the by analysis and that is the residue in the that is acetylated by PCAF and p300 and the of analysis for the of acetylation sites in acetylation sites of p300 and PCAF in HIV-1 Tat we analyzed various the of Tat including a the Tat and in vitro acetylation with p300 the at and in the analysis that to acetyl groups to the the Tat at and the acetylation of the at other amino residues The of the acetylated the signal of the at the of the non-enzymatic acetylation are and are in A. A. of the of of proteins by and mass 2001; Scholar). the acetylation present in the of the reaction that was performed in the of p300 that the transfer of acetyl groups from to the in a non-enzymatic The of after with in the and of p300 was with to of acetyl groups to the as as signal that acetylation of the by p300 in to the non-enzymatic acetylation. of a of Tat that the acetylation of the is the non-enzymatic acetylation of cysteine residues W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar). The of the of acetyl residues in a is for the of acetylation the of which amino other cysteine can non-enzymatic acetylation a amino in an was to in vitro acetylation in the of analysis the of acetyl group to the by a mass of from to a the cysteine residue at acetylation signal at other amino cysteine is to as an acetyl in the of acetyltransferase in in vitro acetylation the analysis of p300 and PCAF acetylation sites in Tat a non-enzymatic acetylation was for the to the Tat and of the with in the of acetyltransferase the signal of the at was in the analysis with the signal of the at the signal of the Tat was the after with in the and of p300 or mass of and of signal after of acetyltransferase to the in vitro acetylation an acetylation of the Tat the Tat for the non-enzymatic acetylation of cysteine was by the the of the and of the and to analysis by a was acetyl mass of was for the of the The mass between the and the is for an of for an the non-enzymatic acetylation of a cysteine residue was by The of in vitro acetylation some the non-enzymatic cysteine the of and the of acetylation sites by the identification of acetylated residues in or proteins the of the non-enzymatic cysteine acetylation can to false-positive acetylation sites are the that lysine is the amino with acetyl in the or protein that a non-enzymatic acetylation are is that the non-enzymatic acetylation of cysteine residues in from amino and W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar). The non-enzymatic cysteine acetylation be considered in of in vitro of or proteins to false-positive the of Tat, the of with is the for the acetylation of the Tat and the of the Tat the non-enzymatic cysteine acetylation was from acetylation that in the Tat is acetylated by PCAF C. L. E. H. F. C. A. Y. M. C. HIV-1 Tat transcriptional activity is regulated by J. 1999; 18: and that is L. Wang D. C. Wang L. H. R. P. Kashanchi F. of the p300 activity by HIV-1 Tat chromatin 2001; Scholar). the of acetyl groups to the and to various by that the acetylation of was to the non-enzymatic acetylation of the cysteine residues present in the W. A. M. M. Acetylation of the HIV-1 Tat an in vitro Chem. 2003; Scholar). be an acetylation in a like the Tat the of cysteine residues the signal in the mass and the of in vitro acetylation are to acetylation in proteins the of substrates is of at 5 amino are for the for the in vitro acetylation reaction be for acetyltransferase the of Tat, acetylation by PCAF was an of amino was used in the in vitro acetylation p300 PCAF was to in an of amino The that the acetylation of by PCAF in Tat that acetylation of a lysine residue by acetyltransferase specific substrate of the including is known that the of a protein domain to the of acetylation sites E. M. K. B. H. vitro acetylation of and proteins by the role of the 2004; Scholar). is in the cell be by the of for in vitro acetylation the of the is with to the and the of the amino the of the analyzed acetylation sites can be and can to example, the acetyltransferase activity of p300 be to in the Tat was by is to the acetylation in after amino using of the techniques for the identification of acetylation sites in or proteins is to an as as to non-enzymatic acetylation of the or protein, to the of the analyzed and to the acetylation amino are performed the analyzed can be by analysis such as or using and cysteine in by analysis using in with or or by analysis using We the for a for for the of Tat and acetylated Tat, for acetyltransferase and for and for for and for analysis of the Tat for the of the and for