The MHC class II (MHCII) processing pathway presents peptides derived from exogenous or membrane-bound proteins to CD4+ T cells. Several studies have shown that glycopeptides are necessary to modulate CD4+ T cell recognition, though glycopeptide structures in these cases are generally unknown. Here, we present a total of 93 glycopeptides from three melanoma cell lines and one matched EBV-transformed line with most found only in the melanoma cell lines. The glycosylation we detected was diverse and comprised 17 different glycoforms. We then used molecular modeling to demonstrate that complex glycopeptides are capable of binding the MHC and may interact with complementarity determining regions. Finally, we present the first evidence of disulfide-bonded peptides presented by MHCII. This is the first large scale study to sequence glyco- and disulfide bonded MHCII peptides from the surface of cancer cells and could represent a novel avenue of tumor activation and/or immunoevasion.Keywords: glycopeptide analysis; immunology; mass spectrometry; MHC class II;

•IIPT reactions using SF6− charge reduce ETD-produced fragment ions.•This spreads ETD fragment ion signals over the entire mass range.•The resulting spectra yield more sequence information and are easier to interpret.•Derivatizations selectively enhance sequence information from the protein termini.•Total sequence coverage for apomyoglobin is 94%.Previously, we described implementation of a front-end ETD (electron transfer dissociation) source for an Orbitrap instrument [1]. This source facilitates multiple fills of the C-trap with product ions from ETD of intact proteins prior to mass analysis. The result is a dramatic enhancement of the observed ion current without the need for time consuming averaging of data from multiple mass measurements. Here we show that ion–ion proton transfer (IIPT) reactions can be used to simplify ETD spectra and to disperse fragment ions over the entire mass range in a controlled manner. We also show that protein derivatization can be employed to selectively enhance the sequence information observed at the N- and C-termini of a protein.

Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT).We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are enriched on nucleosomes containing both active and repressive histone PTMs in human A549 cells and Xenopus embryos.Significantly, we demonstrated that H2A phosphorylation and H4 arginine methylation form a new class of bona fide pre-deposition modifications in the vertebrate embryo. We show that S1ph and R3me containing chromatin domains are not correlated with H3 regulatory PTMs, suggesting a unique role for phosphorylation and arginine methylation.

Electron transfer dissociation (ETD) has improved the mass spectrometric analysis of proteins and peptides with labile post-translational modifications and larger intact masses. Here, the parameters governing the reaction rate of ETD are examined experimentally. Currently, due to reagent injection and isolation events as well as longer reaction times, ETD spectra require significantly more time to acquire than collision-induced dissociation (CID) spectra (>100 ms), resulting in a trade-off in the dynamic range of tandem MS analyses when ETD-based methods are compared to CID-based methods. Through fine adjustment of reaction parameters and the selection of reagents with optimal characteristics, we demonstrate a drastic reduction in the time taken per ETD event. In fact, ETD can be performed with optimal efficiency in nearly the same time as CID at low precursor charge state (z = +3) and becomes faster at higher charge state (z > +3).

Purified histone H2A.Z from chicken erythrocytes and a sodium butyrate-treated chicken erythroleukemic cell line was used as a model system to identify the acetylation sites (K4, K7, K11, K13, and K15) and quantify their distribution in this vertebrate histone variant. To understand the role played by acetylation in the modulation of the H2A.Z nucleosome core particle (NCP) stability and conformation, an extensive analysis was conducted on NCPs reconstituted from acetylated forms of histones, including H2A.Z and recombinant H2A.Z (K/Q) acetylation mimic mutants. Although the overall global acetylation of core histones destabilizes the NCP, we found that H2A.Z stabilizes the NCP regardless of its state of acetylation. Interestingly and quite unexpectedly, we found that the change in NCP conformation induced by global histone acetylation is dependent on H2A/H2A.Z acetylation. This suggests that acetylated H2A variants act synergistically with the acetylated forms of the core histone complement to alter the particle conformation. Furthermore, the simultaneous occurrence of H2A.Z and H2A in heteromorphic NCPs that most likely occurs in vivo slightly destabilizes the NCP, but only in the presence of acetylation.

The activation and recruitment of CD4+ T cells are critical for the development of efficient antitumor immunity and may allow for the optimization of current cancer immunotherapy strategies. Searching for more optimal and selective targets for CD4+ T cells, we have investigated phosphopeptides, a new category of tumor-derived epitopes linked to proteins with vital cellular functions. Although MHC I-restricted phosphopeptides have been identified, it was previously unknown whether human MHC II molecules present phosphopeptides for specific CD4+ T cell recognition. We first demonstrated the fine specificity of human CD4+ T cells to discriminate a phosphoresidue by using cells raised against the candidate melanoma antigen mutant B-Raf or its phosphorylated counterpart. Then, we assessed the presence and complexity of human MHC II-associated phosphopeptides by analyzing 2 autologous pairs of melanoma and EBV-transformed B lymphoblastoid lines. By using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing, and comparative analysis, a total of 175 HLA-DR-associated phosphopeptides were characterized. Many were derived from source proteins that may have roles in cancer development, growth, and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome “fingerprints.” We then generated HLA-DRβ1*0101-restricted CD4+ T cells specific for a phospho-MART-1 peptide identified in both melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen-presenting cells as well as for intact melanoma cells. This previously undescribed demonstration of MHC II-restricted phosphopeptides recognizable by human CD4+ T cells provides potential new targets for cancer immunotherapy.

Histones are highly basic, small proteins that organize eukaryotic DNA into nucleosomes, the building blocks of chromatin. Besides promoting the formation of the chromatin fiber, a large body of work substantiates that histones are intimately involved in gene regulation1, 2. Chromatin-mediated gene regulation is accomplished by post-translational modifications (PTMs) of histones. These PTMs are mostly concentrated on the N-terminal tails that extend beyond the surface of the nucleosome. Prevalent histone PTMs include serine and threonine phosphorylation, arginine methylation (mono- and dimethylation) and lysine acetylation and methylation (mono-, di- and trimethylation). Intriguingly, certain modifications and modification sites have been linked to either gene activation or silencing. Methylation at lysine 9 of histone H3 is known to be located on heterochromatin regions leading to gene repression, whereas methylation of the same histone at lysine 4 is found on actively transcribed genes2. Owing to the excessive amounts of modifications of histones and their observed links to modulating gene expression, theories such as the histone code hypothesis3 have evolved to explain how these modifications can affect cellular events.Traditionally, histone modifications have been discovered and monitored using methods such as site-specific antibodies or 32P-labeling followed by protein sequencing by Edman degradation. However, these techniques are time consuming and have certain limitations that do not enable complete characterization of the myriad of modifications present on histones. Mass spectrometry (MS) has complemented the biological work being conducted on histone PTMs, categorizing novel modifications on histones that were previously not detected by any other means4, 5, 6, 7, 8. Histone proteins pose a formidable challenge to the mass spectrometrist, as their sequences are decorated with an overwhelming number of arginine and lysine residues, especially on the N-termini where most PTMs are known to reside. This causes problems as the most commonly used enzymes usually cleave on either basic or acidic residues. Enzymatic digestion with trypsin results in small peptides that are difficult to retain on RP-HPLC columns and analyzed by MS. On the other hand, proteolysis with enzymes that cleave after acidic residues (e.g., Glu-C) generates larger multiply charged peptides whose MS/MS spectra are difficult if not impossible to interpret. MS-related histone work has benefited from the use of limited trypsin or other enzyme digestion4, 5, 6; however, these methods typically generate several truncated peptides containing the same modification sites, rendering quantification of histone PTMs cumbersome. More recently, the Arg-C digestion of histones has been performed to produce histone peptides that can be used for relative quantification purposes8. Nevertheless, we decided to develop a method that could produce uniform peptides using the robust enzyme trypsin.This method involves the chemical derivatization of histones followed by trypsin digestion resulting in highly reproducible proteolysis fragments that can be quite easily monitored in the mass spectrometer and also allow for a comparative analysis of histone modifications from two distinct cellular conditions9. Our methodology involves derivatizing free amine groups on the N-termini and ε-amino group of unmodified or endogenously monomethylated lysines with propionic anhydride to form propionyl amides before a trypsin cleavage is used. Similar methods using organic acids have also been described, even for in-gel digestion of proteins10, 11, 12. Propionic anhydride derivatization has two practical purposes:1. As all lysine residues will be blocked by the added chemical group or by endogenous modifications, trypsin digestion results in proteolysis only C-terminal to arginine residues mimicking an Arg-C digestion as shown in Figure 1 for histone H3, but with the efficiency and reproducibility of trypsin.2. The derivatization step neutralizes the charge at the N-termini, and unmodified and monomethylated lysine residues. This makes the resulting histone peptides less hydrophilic. They can now be easily resolved by standard RP-HPLC; in addition, the peptides produce doubly and triply charged ions in electrospray ionization MS, and so are pseudo-tryptic-like fragments whose MS/MS spectra are facile to interpret (Fig. 2). This is an advantage over using the Arg-C protease, which will cleave at Arg residues, but not remove charge from lysine residues.As we can now reproducibly generate a set of peptides from one sample to the next regardless of the histone modification status, we can implement a second derivatization step using stable isotope labeling of carboxylic acids for relative quantification and comparison of histone modifications from two separate sources or cellular conditions. The described protocols have been applied for the successful identification and quantification of histone PTMs in cells from various organisms and under various external stimuli13, 14, 15, 16, 17, but can clearly be extended to histones extracted from any cellular source.A typical experimental design for the comparative analysis (i.e., relative quantification) of histone H3 PTMs from two cellular sources is as follows. Histones are acid-extracted from the two different cell samples and purified by HPLC separation into fractions containing distinct family members. The fractions from each sample containing histone H3 are evaporated to dryness and then after reconstitution in a small volume of water are treated with propionic anhydride to block lysine residues. A trypsin digestion is then performed on both samples and another propionic anhydride reaction is performed to modify the newly generated N-termini. Small portions of the two samples are then separately analyzed in the mass spectrometer to determine how much sample (based on total ion current for several peptides) is present from each source. The remaining samples are then treated with methanolic HCl to incorporate a stable isotope label onto the carboxylic acid groups. One sample is treated with “light” methanolic HCl, whereas the other sample is treated with “heavy” methanolic HCl. Roughly equal amounts of sample are mixed (equal amounts judged by previous MS analysis of each sample) and analyzed by MS for a relative comparison of histone PTMs from each cellular state. Typically, these experiments can give information regarding the under- or overexpression of histone PTMs from two different biological conditions. Unmodified histone peptides can serve as controls to determine if equal amounts of each sample were loaded, as their relative abundances should be approximately equal. It is recommended that three replicate runs be performed.Propionic anhydride labeling before digestion, Steps 1–9: 2 hTrypsin digestion and repropionic anhydride, Steps 10–16: 8–12 hStable isotope labeling, Steps 17–22: 3 hMass spectrometry analysis, Steps 23–24: 2 h per sampleData analysis, Steps 25–26: variable, dependent on user Step 24 As several peptides having similar retention times can roughly coelute into the mass spectrometer, undersampling of low-level modified histone peptides is occasionally observed. You may need to adjust the HPLC gradient to make it more gradual to better separate peptides. However, the scan rates on most LTQ ion trap-based mass spectrometers (which we recommend) is more than sufficient to obtain MS/MS spectra for nearly all eluted peptides. Step 25 (incomplete labeling of lysine groups) In Step 5, if the pH drops and you are not able to quickly raise the pH to 8, the result may be incomplete labeling of lysine groups. Unfortunately, inefficient propionylation cannot be assessed until analysis by LC-MS/MS. If a drop in pH is prolonged (< 1 minute), then it is advised to repeat the propionylation step. Step 25 (incomplete labeling of carboxylic groups) In Step 20, if there are trace levels of water in the samples, this could result in incomplete labeling of carboxylic groups. Inefficient conversion to methyl esters can be assessed by LC-MS/MS analysis.

Histone proteins and their accompanying post-translational modifications have received much attention for their ability to affect chromatin structure and, hence, regulate gene expression. Recently, mass spectrometry has become an important complementary tool for the analysis of histone variants and modification sites, for determining the degree of occupancy of these modifications and for quantifying differential expression of these modifications from various samples. Additionally, as advancements in mass spectrometry technologies continue, the ability to read entire ‘histone codes’ across large regions of histone polypeptides or intact protein is possible. As chromatin biology demands, mass spectrometry has adapted and continues as a key technology for the analysis of gene regulation networks involving histone modifications.

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-μg (≈600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or “bivalent domains,” in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1–50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.

A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1–2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the N—Cα bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with the carboxylate anion of benzoic acid. The m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15–40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.

Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.

High-pressure liquid chromatography–tandem mass spectrometry was used to obtain a protein profile of Escherichia coli strain MG1655 grown in minimal medium with glycerol as the carbon source. By using cell lysate from only 3 × 108 cells, at least four different tryptic peptides were detected for each of 404 proteins in a short 4-h experiment. At least one peptide with a high reliability score was detected for 986 proteins. Because membrane proteins were underrepresented, a second experiment was performed with a preparation enriched in membranes. An additional 161 proteins were detected, of which from half to two-thirds were membrane proteins. Overall, 1,147 different E. coli proteins were identified, almost 4 times as many as had been identified previously by using other tools. The protein list was compared with the transcription profile obtained on Affymetrix GeneChips. Expression of 1,113 (97%) of the genes whose protein products were found was detected at the mRNA level. The arithmetic mean mRNA signal intensity for these genes was 3-fold higher than that for all 4,300 protein-coding genes of E. coli. Thus, GeneChip data confirmed the high reliability of the protein list, which contains about one-fourth of the proteins of E. coli. Detection of even those membrane proteins and proteins of undefined function that are encoded by the same operons (transcriptional units) encoding proteins on the list remained low.