Absolute quantitation of post-translational modifications

The cell’s fate is largely governed by the dynamics of cellular events, brought about by the interplay of various proteins, in which post-translational modifications (PTMs) play a key role. Therefore, a better understanding of post-translational modification is of utmost importance to a biochemist. Over the years much work in this regard has been achieved through mass spectrometry, however, the “on-off” nature of PTMs, which accounts for cell’s dynamics, has posed a serious challenge in quantitating them, especially with regards to measuring the occupancy of PTMs on a particular site. Protein phosphorylation has served as the best model for which measurement of occupancy has been attempted by employing stable isotope labeling and label-free approaches. But the reliability of these methods has mainly been limited to the relative quantitation between samples. Although the fractional occupancy/absolute quantitation of phosphorylation has been determined by grossly applying thesemethods, but overall they fall short of testability. Moreover, the field demands a broad approach which can even encompass a more challenging task of absolute quantitation of Cys-based PTMs. For quantitation, differential labeling of samples with stable isotopes or in the label-free approach the peak intensity of a peptide ion or identification frequency of the peptides of a particular protein is used for measurement. Stable isotope labeling is achieved through metabolic labeling in cell culture or by chemical labeling in postmetabolic stage. For the metabolic labeling of proteins, the cell cultures are grown in the presence of naturally abundant amino acids (light version), and amino acids (primarily Leu, Lys, Arg) labeled with heavy isotopes (13C, 15N and/or deuterium), this method is known as stable isotope labeling with amino acids in cell culture (SILAC). The ratio of the ion intensities of the peptide (light vs. heavy) in the MS scan is used for quantitation (Ong et al., 2002). In the post-metabolic labeling approach, such as isotope-coded affinity tag (ICAT) (Sethuraman et al., 2004), isobaric tag for relative and absolute quantitation (iTRAQ) (Gan et al., 2007) and tandemmass tag (TMT) (Thompson et al., 2003), the peptide fragments generated by proteolytic digestion of proteins are chemically derivatized at an amino acid side chain; cysteines thiols (ICAT) or primary amines (iTRAQ and TMT) and quantitation of peptides (and hence the protein) between the samples is done by comparing the ion intensities of the tags in the MS/MS spectrum. Analogous to the SILAC, another approach called Stable isotope labeling in mammals (SILAM) was developed to introduce 15N-label rich diet into a mouse. The ratio of the ion intensities of 14N (light)/15N (heavy) peptides was used to check the changes in the protein expression in tissues (Wu et al., 2004; McClatchy et al., 2007), differential regulation of phosphorylation site within a protein (Tao et al., 2009) or in quantitative phosphoproteomics (Ficarro et al., 2002). The rationale behind SILAC and SILAM approaches is to counteract the potential problem of systematic errors associated in post-metabolic labeling strategy. Here, the light and heavy samples are mixed after the sample preparation, wherein, in the metabolic labeling approach, the translational machinery of the cell incorporates heavy label into the whole proteome, and the light and heavy samples are mixed prior to sample preparation. Stable isotope labeling for absolute quantitation requires internal standards that are used to measure protein abundance per se. These standards are the pre-selected synthetic peptides from the protein of interest that gets labeled with an isotope. A known amount of the isotope labeled peptides are mixed with the sample and then introduced into the mass spectrometer. Absolute abundance of the PTM can be obtained by measuring the relative abundance of the modified and unmodified peptides and comparing it with the amount of standard used. Notably, stable isotope labeling and synthetic peptide standards were used in absolute quantitation of phosphorylation on Ser1526 in human separase (Stemmann et al., 2001) and phosphorylation in Akt (Atrih et al., 2010). Moreover, the SILAC approach was used to quantitate global phosphoproteomics during mitosis (Olsen et al., 2010). These methods have set the base for absolute quantitation, but they are inefficient and error-prone. The stable isotope labeling approach is biased, and the synthesis of peptide standards is costly, time-intensive and, may induce run-to-run variation in LC-MS/MS analysis. Also, the use of internal standards becomes unreliable when a pathophysiologic condition gives rise to a (new) site which is not included in the internal standards. Label-free approach is another way to quantify but in this technique, there is a common assumption that the ion intensities of the modified and unmodified peptides (peptides derived from the same protein) are the same which probably is not the case. Therefore, we need