Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry

Ambrosius P. L. Snijders 0 2 Ming-Lung Hung 0 1 Stuart A. Wilson 0 1 Mark J. Dickman 0 2 0 Address reprint requests to Dr. M. J. Dickman, Department of Chemical and Process Engineering, University of Sheffield , Sheffield S3 7RD, UK 1 Department of Molecular Biology and Biotechnology, University of Sheffield , Sheffield, United Kingdom 2 Biological and Environmental Systems, Department of Chemical and Process Engineering, University of Sheffield , Sheffield, United Kingdom Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches. (J Am Soc Mass Spectrom 2010, 21, 88 -96) 2010 American Society for Mass Spectrometry - Atein post-translational modifications (PTMs) inrginine and lysine methylation are common provolved in transcriptional regulation, DNA repair, RNA processing, and signal transduction [1 4]. With the recent identification of enzymes capable of demethylating lysine and arginine residues [57], and the observation that arginine methylation on histones can be antagonized enzymatically (via deimination of arginine to citrulline) [8, 9], it seems likely that methylation can contribute to the dynamic control of biological processes [10]. Protein arginine N-methyltransferases (PRMTs) catalyze the post-translational transfer of a methyl group from the donor S-adensoyl-L-methioinine (SAM) to arginine residues [4, 11]. Three forms of arginine methylation have been described NG-monomethylarginine, NGNG-dimethylarginine (asymmetric dimethylarginine aDMA), and NGN=Gdimethylarginine (symmetric dimethylarginine sDMA). Lysine can be progressively methylated by lysine or histone methyltransferases to give -N-monomethyllysine, -N-dimethyllysine, or -N-trimethyllysine. In addition to lysine methylation in eukaryotes, it is increasingly clear that lysine methylation is abundant throughout the prokaryotic kingdom [1214]. Traditionally, protein methylation is detected by Edman sequencing, radioactively by using the tritiated methyltransferase cofactor SAM or via immuno-detection using methylation specific antibodies. Arginine dimethyl antibodies have also been used to enrich for arginine dimethylated proteins [15]. Unfortunately many of these approaches are either aspecific or fail to identify the site and the type of methylation. Mass spectrometry has become the main analytical tool in protein identification and also in the discovery and characterization of PTMs. In some cases, the characteristic fragmentation properties of peptides containing PTMs, such as neutral losses or specific reporter masses, can be exploited. Precursor ion scanning is used to study arginine methylation since two side-chain fragments of DMA, the dimethylammonium ion (46.06 Da) and dimethylcarbodiimidium ions (71.06 Da) can be used as specific reporters for arginine methylation [16 18]. Dimethylcarbodiimidium is produced from both aDMA and sDMA, but generally more strongly for sDMA. In addition, side-chain fragmentation of arginine methylated peptides can often be observed as neutral losses in MS/MS spectra and therefore can be used to determine the type of methylation. A neutral loss of monomethylamine (31.04 Da) is specific for MMA and sDMA, dimethylamine (45.05 Da) for aDMA, and dimethylcarbodiimidium (70.05 Da) for aDMA and sDMA [18, 19]. Precursor ion scanning is also used to detect the immonium ions of lysine mono- and dimethylation (98.1, 112.1 Da). These approaches have been extensively applied for the characterization of histone PTMs [20, 21]. Despite the development of these approaches, standard LC-MS workflows for arginine and lysine methylated peptides still suffer from a number of disadvantages. Arginine and lysine methylation lead to missed trypsin cleavage sites and arginine methylation predominantly occurs in conserved glycine/arginine rich sequences called GAR or RGG motifs [22, 23]. Methylated peptides therefore frequently contain internal hydrophilic residues, which makes them difficult to capture on hydrophobic stationary phases employed in reversed-phase LC-MS. Moreover, peptides with internal basic residues often generate poor collision induced (CID) spectra. This is most likely because basic residues sequester protons, thereby reducing their mobility along the peptide backbone and preventing dissociation. As described above, side-chain fragmentation can be exploited for diagnostic purposes. However, reduced fragmentation along the peptide backbone will ultimately compromise the amount of sequence information that can be obtained from such spectra. Electron-transfer dissociation (ETD) is an alternative fragmentation technique related to electron capture dissociation (ECD) that promotes cleavage of NC bonds in the peptide backbone resulting in fragment ions of the type c= and z= [24]. In general, ETD is particularly effective for highly charged peptides and is less susceptible to side-chain fragmentation compared with CID. Therefore, ETD has become a popular method to study labile post-translational modifications, such as phosphorylation, glycosylation, nitrosylation, sulfonation etc. [25]. ETD and ECD have been extensively used in the study of histone modifications [26 29]. Using these techniques, it is possible to obtain extensive sequence information on large peptides and even intact proteins in an approach referred to as top-down sequencing [26 29]. Recently, ETD was (...truncated)