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Table S1. Summary of samples used for this study. Table S2. Antibodies used in the study. Figure S1. Mass spectra of all peptides referenced in figures 2 and 3. Figure S2. ETD Mass spectrum of RPPItQSSL containing an asymmetrically dimethylated Arg residue. Figure S3. ICS flow cytometry plots for HD5. Figure S4. Gating strategy for phenotyping shown in Fig 2E. Figure S5. Healthy donor immunity to O-GlcNAc peptides measured using IFNgamma ELISpot. Figure S6. ICS flow cytometry plots for HD5. Figure S7. Positional analysis of O-GlcNAc peptides.

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.

We provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Chemical proteomics is widely used for the global investigation of protein activity and binding of small molecule ligands. Covalent probe binding and inhibition are assessed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to gain molecular information on targeted proteins and probe-modified sites. The identification of amino acid sites modified by large complex probes, however, is particularly challenging because of the increased size, hydrophobicity, and charge state of peptides derived from modified proteins. These studies are important for direct evaluation of proteome-wide selectivity of inhibitor scaffolds used to develop targeted covalent inhibitors. Here, we disclose reverse-phase chromatography and MS dissociation conditions tailored for binding site identification using a clickable covalent kinase inhibitor containing a sulfonyl-triazole reactive group (KY-26). We applied this LC-MS/MS strategy to identify tyrosine and lysine sites modified by KY-26 in functional sites of kinases and other ATP-/NAD-binding proteins (>65 in total) in live cells. Our studies revealed key bioanalytical conditions to guide future chemical proteomic workflows for direct target site identification of complex irreversible probes and inhibitors.

Complete sequence coverage of monospecific antibodies was previously achieved using immobilized aspergillopepsin I in a single LC-MS/MS analysis. Bispecific antibodies are asymmetrical compared to their monospecific antibody counterparts, resulting in a decrease in the concentration of individual subunits. Four standard proteins were used to characterize the effect of a decrease in concentration when using this immobilized enzyme reactor. Low concentration samples resulted in the elimination of large peptide products due to a greater number of enzymatic cleavages. A competitive inhibitor rich in arginine residues reduced the number of enzymatic cleavages to the protein and retained large molecular weight products. The digestion of a bispecific antibody with competitive inhibition of aspergillopepsin I maintained large peptide products better suited for sequence reconstruction, resulting in complete sequence coverage from a single LC-MS/MS analysis.

RNA granules are cytoplasmic condensates that organize biochemical and signaling complexes in response to cellular stress. Functional proteomic investigations under RNA-granule-inducing conditions are needed to identify protein sites involved in coupling stress response with ribonucleoprotein regulation. Here, we apply chemical proteomics using sulfonyl-triazole (SuTEx) probes to capture cellular responses to oxidative and nutrient stress. The stress-responsive tyrosine and lysine sites detected mapped to known proteins involved in processing body (PB) and stress granule (SG) pathways, including LSM14A, FUS, and Enhancer of mRNA-decapping protein 3 (EDC3). Notably, disruption of EDC3 tyrosine 475 (Y475) resulted in hypo-phosphorylation at S161 and S131 and altered protein-protein interactions (PPIs) with decapping complex components (DDX6, DCP1A/B) and 14-3-3 proteins. This resulting mutant form of EDC3 was capable of rescuing the PB-deficient phenotype of EDC3 knockout cells. Taken together, our findings identify Y475 as an arsenic-responsive site that regulates RNA granule formation by coupling EDC3 post-translational modification and PPI states.

Protamines are small, highly-specialized, arginine-rich, and intrinsically-disordered chromosomal proteins that replace histones during spermiogenesis in many organisms. Previous evidence supports the notion that, in the animal kingdom, these proteins have evolved from a primitive replication-independent histone H1 involved in terminal cell differentiation. Nevertheless, a direct connection between the two families of chromatin proteins is missing. Here, we primarily used electron transfer dissociation MS-based analyses, revealing that the protamines in the sperm of the liverwort Marchantia polymorpha result from post-translational cleavage of three precursor H1 histones. Moreover, we show that the mature protamines are further post-translationally modified by di-aminopropanelation, and previous studies have reported that they condense spermatid chromatin through a process consisting of liquid-phase assembly likely involving spinodal decomposition. Taken together, our results reveal that the interesting evolutionary ancestry of protamines begins with histone H1 in both the animal and plant kingdoms.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion’s unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

We have enabled parallel ion parking on a modified Orbitrap Elite™ as a way to control ion-ion proton transfer reactions via selective activation of a range of ions. The result is the concentration of the majority of ion current from multiple charge states of each precursor proteoform into a single charge state, maximizing signal intensity and increasing effective sensitivity compared to conventional MS1 spectra. These techniques were applied in an on-line HPLC, data-dependent MS/MS analysis of intact E. coli ribosomal proteins with HCD fragmentation. With one injection, all but two ribosomal proteins were selected for fragmentation and subsequently identified. The techniques described facilitate rapid identification of intact proteins in complex mixtures and an enhanced ability to observe proteins of low abundance., Graphical Abstract

International audience; The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (∼0.1% of total arginine), Rme2a was highly abundant (∼1.1%), and Rme2s was intermediate (∼0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.

Summary Protein arginine methyltransferases (PRMTs) catalyze the post-translational monomethylation (Rme1), asymmetric (Rme2a), or symmetric (Rme2s) dimethylation of arginine. To determine the cellular consequences of type I (Rme2a) and II (Rme2s) PRMTs, we developed and integrated multiple approaches. First, we determined total cellular dimethylarginine levels, revealing that Rme2s was ∼3% of total Rme2 and that this percentage was dependent upon cell type and PRMT inhibition status. Second, we quantitatively characterized in vitro substrates of the major enzymes and expanded upon PRMT substrate recognition motifs. We also compiled our data with publicly available methylarginine-modified residues into a comprehensive database. Third, we inhibited type I and II PRMTs and performed proteomic and transcriptomic analyses to reveal their phenotypic consequences. These experiments revealed both overlapping and independent PRMT substrates and cellular functions. Overall, this study expands upon PRMT substrate diversity, the arginine methylome, and the complex interplay of type I and II PRMTs., Graphical abstract, Highlights • Rapid and sensitive direct-injection MS/MS dimethylarginine quantification • Quantitative methyltransferase assays on oriented peptide array libraries (OPALs) • PTMScan, proteomic, transcriptomic, and phenotypic analysis of PRMT inhibition • Compilation of our data and publicly available arginine methylome data, Molecular biology; Cell biology

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376–84. ©2017 AACR.

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.

The centromere is the locus on the chromosome that acts as the essential connection point between the chromosome and the mitotic spindle. A histone H3 variant, CENP-A, defines the location of the centromere, but centromeric chromatin consists of a mixture of both CENP-A-containing and H3-containing nucleosomes. We report a surprisingly uniform pattern of primarily monomethylation on lysine 20 of histone H4 present in short polynucleosomes mixtures of CENP-A and H3 nucleosomes isolated from functional centromeres. Canonical H3 is not a component of CENP-A-containing nucleosomes at centromeres, so the H3 we copurify from these preparations comes exclusively from adjacent nucleosomes. We find that CENP-A-proximal H3 nucleosomes are not uniformly modified but contain a complex set of PTMs. Dually modified K9me2-K27me2 H3 nucleosomes are observed at the centromere. Side-chain acetylation of both histone H3 and histone H4 is low at the centromere. Prior to assembly at centromeres, newly expressed CENP-A is sequestered for a large portion of the cell cycle (late S-phase, G2, and most of mitosis) in a complex that contains its partner, H4, and its chaperone, HJURP. In contrast to chromatin associated centromeric histone H4, we show that prenucleosomal CENP-A-associated histone H4 lacks K20 methylation and contains side-chain and α-amino acetylation. We show HJURP displays a complex set of serine phosphorylation that may potentially regulate the deposition of CENP-A. Taken together, our findings provide key information regarding some of the key components of functional centromeric chromatin.

Phosphorylation events within cancer cells often become dysregulated, leading to oncogenic signaling and abnormal cell growth. Phosphopeptides derived from aberrantly phosphorylated proteins that are presented on tumors and not on normal tissues by human leukocyte antigen (HLA) class I molecules are promising candidates for future cancer immunotherapies, because they are tumor specific and have been shown to elicit cytotoxic T cell responses. Robust phosphopeptide enrichments that are suitable for low input amounts must be developed to characterize HLA-associated phosphopeptides from clinical samples that are limited by material availability. We present two complementary mass spectrometry–compatible, iron(III)-immobilized metal affinity chromatography (IMAC) methods that use either nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) in-house-fabricated columns. We developed these protocols to enrich for subfemtomole-level phosphopeptides from cell line and human tissue samples containing picograms of starting material, which is an order of magnitude less material than what is commonly used. In addition, we added a peptide esterification step to increase phosphopeptide specificity from these low-input samples. To date, hundreds of phosphopeptides displayed on melanoma, ovarian cancer, leukemia and colorectal cancer have been identified using these highly selective phosphopeptide enrichment protocols in combination with a program called ‘CAD Neutral Loss Finder’ that identifies all spectra containing the characteristic neutral loss of phosphoric acid from phosphorylated serine and threonine residues. This methodology enables the identification of HLA-associated phosphopeptides presented by human tissue samples containing as little as nanograms of peptide material in 2 d.

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

Centromeres are chromosomal loci required for accurate segregation of sister chromatids during mitosis. The location of the centromere on the chromosome is not dependent on DNA sequence, but rather it is epigenetically specified by the histone H3 variant centromere protein A (CENP-A). The N-terminal tail of CENP-A is highly divergent from other H3 variants. Canonical histone N termini are hotspots of conserved posttranslational modification; however, no broadly conserved modifications of the vertebrate CENP-A tail have been previously observed. Here, we report three posttranslational modifications on human CENP-A N termini using high-resolution MS: trimethylation of Gly1 and phosphorylation of Ser16 and Ser18. Our results demonstrate that CENP-A is subjected to constitutive initiating methionine removal, similar to other H3 variants. The nascent N-terminal residue Gly1 becomes trimethylated on the α-amino group. We demonstrate that the N-terminal RCC1 methyltransferase is capable of modifying the CENP-A N terminus. Methylation occurs in the prenucleosomal form and marks the majority of CENP-A nucleosomes. Serine 16 and 18 become phosphorylated in prenucleosomal CENP-A and are phosphorylated on asynchronous and mitotic nucleosomal CENP-A and are important for chromosome segregation during mitosis. The double phosphorylation motif forms a salt-bridged secondary structure and causes CENP-A N-terminal tails to form intramolecular associations. Analytical ultracentrifugation of phospho-mimetic CENP-A nucleosome arrays demonstrates that phosphorylation results in greater intranucleosome associations and counteracts the hyperoligomerized state exhibited by unmodified CENP-A nucleosome arrays. Our studies have revealed that the major modifications on the N-terminal tail of CENP-A alter the physical properties of the chromatin fiber at the centromere.

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).

Tandem mass spectra (MS/MS) produced using electron transfer dissociation (ETD) differ from those derived from collision-activated dissociation (CAD) in several important ways. Foremost, the predominant fragment ion series are different: c- and z(*)-type ions are favored in ETD spectra while b- and y-type ions comprise the bulk of the fragments in CAD spectra. Additionally, ETD spectra possess charge-reduced precursors and unique neutral losses. Most database search algorithms were designed to analyze CAD spectra, and have only recently been adapted to accommodate c- and z(*)-type ions; therefore, inclusion of these additional spectral features can hinder identification, leading to lower confidence scores and decreased sensitivity. Because of this, it is important to pre-process spectral data before submission to a database search to remove those features that cause complications. Here, we demonstrate the effects of removing these features on the number of unique peptide identifications at a 1% false discovery rate (FDR) using the open mass spectrometry search algorithm (OMSSA). When analyzing two biologic replicates of a yeast protein extract in three total analyses, the number of unique identifications with a approximately 1% FDR increased from 4611 to 5931 upon spectral pre-processing--an increase of approximately 28.6%. We outline the most effective pre-processing methods, and provide free software containing these algorithms.

Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. 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 fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. m / z values for the resulting singly and doubly charged ions are used to read a sequence of 15–40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.

Methodology for sequence analysis of ∼150 kDa monoclonal antibodies (mAb), including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured (reduced and alkylated) antibody was accomplished in seconds by flowing a sample in 8 m urea at a controlled flow rate through a micro column reactor containing immobilized aspergillopepsin I. The resulting product mixture containing 3–9 kDa peptides was then fractionated by capillary column liquid chromatography and analyzed on-line by both electron-transfer dissociation and collisionally activated dissociation mass spectrometry (MS). This approach enabled identification of peptides that cover the complete sequence of a murine mAb. With customized tandem MS and ProSightPC Biomarker search, we verified 95% amino acid residues of this mAb and identified numerous post-translational modifications (oxidized methionine, pyroglutamylation, deamidation of Asn, and several forms of N-linked glycosylation). For disulfide bond location, native mAb is subjected to the same procedure but with longer digestion times controlled by sample flow rate through the micro column reactor. Release of disulfide containing peptides from accessible regions of the folded antibody occurs with short digestion times. Release of those in the interior of the molecule requires longer digestion times. The identity of two peptides connected by a disulfide bond is determined using a combination of electron-transfer dissociation and ion–ion proton transfer chemistry to read the two N-terminal and two C-terminal sequences of the connected peptides.

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.

Experimental allergic encephalomyelitis (EAE) is an animal model for multiple sclerosis induced by stimulating myelin basic protein (MBP)-specific T cells. The MBP-specific repertoire in B10.PL mice is shaped by tolerance mechanisms that eliminate MBP121–150–specific T cells. In contrast, MBPAc1–11–specific T cells escape tolerance and constitute the encephalitogenic repertoire. To determine if this differential tolerance is caused by differences in the abundance of MBP epitopes generated by processing, MBP peptides were eluted from I-Au complexes and analyzed by mass spectrometry. Peptides were identified from both the NH2-terminal and MBP121–150 regions. Unexpectedly, MBPAc1–18 and Ac1–17, which contain the MBPAc1–11 epitope, were much more abundant than MBP121–150 peptides. The results demonstrate that competition between two I-Au binding registers, a low affinity register defined by MBPAc1–11 and a high affinity register defined by MBP5–16, prevents most of the NH2-terminal naturally processed peptides from binding in the MBPAc1–11 register. The small fraction of MBPAc1–18 bound in the MBPAc1–11 register is not sufficient to induce tolerance but provides a ligand for MBPAc1–11–specific T cells during disease. These results provide a basis for both the lack of tolerance to MBPAc1–11 and the ability of this epitope to become a target during autoimmunity.

We created a web-based tutorial designed to teach manual interpretation and identification of spectra acquired using electron transfer dissociation (ETD). The tutorial provides an explanation of the ETD fragmentation process with the goal of identifying all of the significant peaks in a spectrum. We discuss determination of the precursor mass and charge state, neutral losses, electron transfer without dissociation (ETnoD), and the mechanisms by which fragment ions are created. Our hope is to provide a tool that presents the information already taught in D.F.H.’s short courses in a way that is easy for any student or researcher in the mass spectrometry community to access. The tutorial may be found at http://www.huntlab.org.

Background Electron Transfer Dissociation [ETD] can dissociate multiply charged precursor polypeptides, providing extensive peptide backbone cleavage. ETD spectra contain charge reduced precursor peaks, usually of high intensity, and whose pattern is dependent on its parent precursor charge. These charge reduced precursor peaks and associated neutral loss peaks should be removed before these spectra are searched for peptide identifications. ETD spectra can also contain ion-types other than c and z˙. Modifying search strategies to accommodate these ion-types may aid in increased peptide identifications. Additionally, if the precursor mass is measured using a lower resolution instrument such as a linear ion trap, the charge of the precursor is often not known, reducing sensitivity and increasing search times. We implemented algorithms to remove these precursor peaks, accommodate new ion-types in noise filtering routine in OMSSA and to estimate any unknown precursor charge, using Linear Discriminant Analysis [LDA]. Results Spectral pre-processing to remove precursor peaks and their associated neutral losses prior to protein sequence library searches resulted in a 9.8% increase in peptide identifications at a 1% False Discovery Rate [FDR] compared to previous OMSSA filter. Modifications to the OMSSA noise filter to accommodate various ion-types resulted in a further 4.2% increase in peptide identifications at 1% FDR. Moreover, ETD spectra when searched with charge states obtained from the precursor charge determination algorithm is shown to be up to 3.5 times faster than the general range search method, with a minor 3.8% increase in sensitivity. Conclusion Overall, there is an 18.8% increase in peptide identifications at 1% FDR by incorporating the new precursor filter, noise filter and by using the charge determination algorithm, when compared to previous versions of OMSSA.

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

We describe the design and performance of a prototype high performance hybrid mass spectrometer. This instrument consists of a linear quadrupole ion trap (QLT) coupled to a Fourier transform ion cyclotron resonance mass analyzer (FTMS). This configuration provides rapid and automated MS and MS/MS analyses, similar to the "data dependent scanning" found on standard 3-D Paul traps, but with substantially improved internal scan dynamic range, mass measurement accuracy, mass resolution, and detection limits. Sequence analysis of peptides at the zeptomole level is described. The recently released, commercial version of this instrument operates in the LC/MS mode (1 s/scan) with a mass resolution of 100 000 and is equipped with automatic gain control to provide mass measurement accuracy of 1-2 ppm without internal standard. Methodology is described that uses this instrument to compare the post-translational modifications present on histone H3 isolated from asynchronously growing cells and cells arrested in mitosis.

The principal objective of this experiment was to investigate whether previous reports of a relation between locomotor status and stage 4 object permanence performance generalized to performance on a different object localization task. A secondary objective was to evaluate the contribution of visual tracking as a mediating variable in the relation between locomotor experience and search performance. Precrawling (n= 20), crawling (n= 10), and creeping (n= 18) infants (M= 33 weeks old) were tested using a search task in which either they or the hiding containers were rotated 180° before search was permitted. The results revealed that locomotor status was related to search performance following a displacement of the infant, but not following a displacement of the containers. Moreover, visual tracking of the correct container was not related to locomotor status, even though it was related to search performance. These findings suggest that the effects of locomotor experience on infants' search performance are quite specific and are mediated by a variety of factors that do not necessarily generalize across search tasks.

An integrated software package for testing infants in a habituation procedure is described. The software is written for an Apple II computer with a minimum of 64K of memory. Additional interface cards are required for timing responses and displaying dynamic stimuli. The software is divided into four independent modules: (1) Configure Experiment allows the experimenter to design a specific experiment. (2) Run Experiment controls the running of a previously configured habituation experiment. (3) Calculate Reliability allows a second observer to score the subject for the purpose of computing reliability. (4) Data Summary and Formatting provides a summary of the results and allows the user to flexibly format the data for reading by various statistical packages.