Characterization Of Post-translational Modifications Research Articles

Data Processing Algorithms for Analysis of High Resolution MSMS Spectra of Peptides with Complex Patterns of Posttranslational Modifications

The emergence of efficient fragmentation methods such as electron capture dissociation (ECD) and electron transfer dissociation (ETD) provides the opportunity for detailed structural characterization of heavily covalently modified large peptides and small proteins such as intact histones. Even with effective gas phase ion isolation so that a single molecular precursor ion is selected, the MSMS spectrum of a heavily modified peptide may reveal the presence of a mixture of peptides with the same amino acid sequence and the same total number of posttranslational modification (PTM) moieties (same PTM composition) but with different PTM configurations or site-specific occupancy isoforms. Currently available data analysis methods depend on a deisotoping procedure, which becomes less effective when spectra (fragmentation patterns) contain many overlapping isotopic distributions. Peptide database search engines can only identify the most abundant PTM configuration (PTM arrangement on different residues) in such mixtures. To identify all the PTM configurations present in these mixtures and to estimate their relative abundances, we extended our fragment assignment by visual assistance program to search for ions representing all possible configurations, subjected to the total PTM composition constraint. This resulted in the identification of PTM configurations supported by unique fragment ions, and their relative abundances were estimated by use of a non-negative least squares procedure.

Challenges in Computational Analysis of Mass Spectrometry Data for Proteomics

Mass spectrometry is an analytical technique for determining the composition of a sample. Recently it has become a primary tool for protein identification and quantification, and post translational modification characterization in proteomics research. Both the size and the complexity of the data produced by this experimental technique impose great computational challenges in the data analysis. This article reviews some of these challenges and serves as an entry point for those who want to study the area in general.

Towards Liquid Chromatography Time-Scale Peptide Sequencing and Characterization of Post-Translational Modifications in the Negative-Ion Mode Using Electron Detachment Dissociation Tandem Mass Spectrometry

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.

Strategy for Comprehensive Identification of Post-translational Modifications in Cellular Proteins, Including Low Abundant Modifications: Application to Glyceraldehyde-3-phosphate Dehydrogenase

Post-translational modifications (PTMs) play key roles in the regulation of biological functions of proteins. Although some progress has been made in identifying several PTMs using existing approaches involving a combination of affinity-based enrichment and mass spectrometric analysis, comprehensive identification of PTMs remains a challenging problem in proteomics because of the dynamic complexities of PTMs in vivo and their low abundance. We describe here a strategy for rapid, efficient, and comprehensive identification of PTMs occurring in biological processes in vivo. It involves a selectively excluded mass screening analysis (SEMSA) of unmodified peptides during liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-q-TOF MS/MS) through replicated runs of a purified protein on two-dimensional gel. A precursor ion list of unmodified peptides with high mass intensities was obtained during the initial run followed by exclusion of these unmodified peptides in subsequent runs. The exclusion list can grow as long as replicate runs are iteratively performed. This enables the identifications of modified peptides with precursor ions of low intensities by MS/MS sequencing. Application of this approach in combination with the PTM search algorithm MODi to GAPDH protein in vivo modified by oxidative stress provides information on multiple protein modifications (19 types of modification on 42 sites) with >92% peptide coverage and the additional potential for finding novel modifications, such as transformation of Cys to Ser. On the basis of the information of precursor ion m/z, quantitative analysis of PTM was performed for identifying molecular changes in heterogeneous protein populations. Our results show that PTMs in mammalian systems in vivo are more complicated and heterogeneous than previously reported. We believe that this strategy has significant potential because it permits systematic characterization of multiple PTMs in functional proteomics.

Using Mass Spectrometry to Characterize the Complex Posttranslational Modifications of Histones

As one of the major molecular mechanisms that mediate epigenetics, histone proteins undergo diverse posttranslational modifications (PTMs), most notably acetylation and methylation. It is now well known that different PTMs can co-occur on proteins that may act sequentially or in combination and constitute a ‘histone code’. Mass spectrometry is the method of choice for studying protein PTMs. Conventional mass spectrometry strategy relies on protein digest analysis using chromatographic separation of peptides and tandem mass spectrometry with collisional induced dissociation (CID) for fragmentation analysis. Although usually sensitive enough to determine even low stoichiometry modifications, it is difficult to determine the co-occurrence of different modifications on the same protein species using this analytical approach. However, new fragmentation mechanisms such as electron capture dissociation (ECD) are capable of preserving labile PTMs and allow characterization of PTMs at the intact protein level. Through our study of histone modifications from several different samples, this presentation will show the power of ECD for protein modification analysis. Specific PTM patterns will be compared among different histone preparations to further the understanding of how epigenetic regulation can be modulated through histone modification. Financial support was provided by NCRR 01614(ALB), NIH R01 GM63959 (CDA) and NSF CHE-99-09502 (AGM).

Precise proteomic identification using mass spectrometry coupled with stable isotope labeling

Stable isotope labeling (SIL) can assist mass spectrometry to improve its specificity and throughput in protein identification, de novo sequencing, and characterization of post translational modifications. This Education article summarizes the unique characteristics of stable isotope-assisted mass spectrometry for accurate protein identification without requirements for ultrahigh mass accuracy. Some applications are discussed here to demonstrate the general experimental procedures, data interpretation, and the improvements in accuracy and throughput compared to the use of mass spectrometry alone.

Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage.

A comprehensive approach to protein identification and determination of sites of posttranslational modifications (PTMs) in heavily modified proteins was tested. In this approach, termed "reconstructed molecular mass analysis" (REMMA), the molecular mass distribution of the intact protein is measured first, which reveals the extent and heterogeneity of modifications. Then the protein is digested with one or several enzymes, with peptides separated by reversed-phase HPLC, and analyzed by Fourier transform mass spectrometry (FTMS). Vibrational excitation (collisional or infrared) or electron capture dissociation (ECD) of peptide ions provides protein identification. When a measured peptide molecular mass indicates the possibility of a PTM, vibrational excitation is applied to determine via characteristic losses the type and eventually the structure of the modification, while ECD determines the PTM site. Chromatographic peak analysis continues until full sequence coverage is obtained, after which the molecular mass is reconstructed and compared with the measured value. An agreement indicates that the PTM characterization was complete. This procedure applied to the bovine milk PP3 protein containing 25% modifications by weight yielded all known modifications (five phosphorylations, two O- and one N-glycosylation) as well as the previously unreported NeuNAc-Hex-[NeuNAc]HexNAc group O-linked to Ser60. With the FTMS performance improved, REMMA can serve as the basis for high-throughput, high-sensitivity PTM characterization of biological important proteins, which should speed up the proteomics research.

2436 Angiotensinergic circuits associated noradrenaline release in the median preoptic area in rats

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.