High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.The repertoires and levels of peptides, presented by the major histocompatibility complex (MHC)¹ class I molecules at the cells'' surface, are modulated by multiple factors. These include the rates of synthesis and degradation of their source proteins, the transport efficacy of the peptides through the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), their subsequent processing and loading onto the MHC molecules within the ER, and the rates of transport of the MHC molecules with their peptide cargo to the cell surface. The off-rates of the presented peptides, the residence time of the MHC complexes at the cell surface, and their retrograde transport back into the cytoplasm, influence, as well, the presented peptidomes (reviewed in (1)). Even though significant portions of the MHC class I peptidomes are thought to be derived from newly synthesized proteins, including misfolded proteins, defective ribosome products (DRiPs), and short lived proteins (SLiPs), most of the MHC peptidome is assumed to originate from long-lived proteins, which completed their functional cellular roles or became defective (retirees), (reviewed in (2)).The main protease, supplying the MHC peptidome production pipeline, is thought to be the proteasome (3). It is also responsible for generation of the final carboxyl termini of the MHC peptides (4), (reviewed in (5)). The final trimming of the n-termini of the peptides, within the endoplasmic reticulum (ER), is thought to be performed by amino peptidases, such as ERAP1/ERAAP, which fit the peptides into their binding groove on the MHC molecules (6) (reviewed in (7)). Nonproteasomal proteolytic pathways were also suggested as possible contributors to the MHC peptidome, including proteolysis by the ER resident Signal peptide peptidase (8, 9), the cytoplasmic proteases Insulin degrading enzyme (10), Tripeptidyl peptidase (11–13), and a number of proteases within the endolysosome pathway (reviewed recently in (14–17)). In contrast to the mostly cytoplasmic and ER production of the MHC class I peptidome, the class II peptidome is produced in a special compartment, associated with the endolysosome pathway (18–20). This pathway is also thought to participate in the cross presentation of class I peptides, derived from proteins up-taken by professional antigen presenting cells (21), (reviewed in (15–17, 22)).The centrality of the proteasomes in the generation of the MHC peptidome was deduced mostly from the observed change in presentation levels of small numbers of selected peptides, following proteasome inhibition (3, 23). Even the location of some of the genes encoding the catalytic subunits of the immunoproteasome (LMP2 and LMP7) (24) within the MHC class II genomic locus, was suggested to support the involvement of the proteasome in the generation of the MHC class I peptidome (25). Similar conclusions were deduced from alterations in peptide presentation, following expression of the catalytic subunits of the immunoproteasome (26), (reviewed in (5)). Yet, although most of the reports indicated reductions in presentation of selected peptides by proteasome inhibition (3, 27–29), others have observed only limited, and sometimes even opposite effects (23, 30–32).The matter is further complicated by the indirect effects of proteasome inhibition used for such studies on the arrest of protein synthesis by the cells (33–35), on the transport rates of the MHC molecules to the cell surface, and on their retrograde transport back to the vesicular system (36) (reviewed in (37)). Proteasome inhibition likely causes shortage of free ubiquitin, reduced supply of free amino acids, and induces an ER unfolded protein response (UPR), which signals the cells to block most (but not all) cellular protein synthesis (reviewed in (38)). Because a significant portion of the MHC peptidome originates from degradation of DRiPs and SLiPs (reviewed in (2)), arrest of new protein synthesis should influence the presentation of their derived MHC peptides. Taken together, these arguments may suggest that merely following the changes in the presentation levels of the MHC molecules, or even of specific MHC peptides, after proteasome inhibition, does not provide the full picture for deducing the relative contribution of the proteasomal pathway to the production of the MHC peptidome (reviewed in (7)).MHC peptidome analysis can be performed relatively easily by modern capillary chromatography combined with mass spectrometry (reviewed in (39)). The peptides are recovered from immunoaffinity purified MHC molecules after detergent solubilization of the cells (40, 41), from soluble MHC molecules secreted to the cells'' growth medium (42, 43) or from patients'' plasma (44). The purified peptides pools are resolved by capillary chromatography and the individual peptides are identified and quantified by tandem mass spectrometry (40), (reviewed in (45–47)). In cultured cells, quantitative analysis can also be followed by metabolic incorporation of stable isotope labeled amino acids (SILAC) (48). Furthermore, the rates of de novo synthesis of both MHC peptides and their proteins of origin can be followed using the dynamic-SILAC proteomics approach (49) with its further adaptation to HLA peptidomics (50–52).This study attempts to define the relative contribution of the proteasomes to the production of HLA class I peptidome by simultaneously following the effects of proteasome inhibitors, epoxomicin and bortezomib (Velcade), on the rates of de novo synthesis of both the HLA class I peptidome and the cellular proteome of the same MCF-7 human breast cancer cultured cells. The proteasome inhibitors did not reduce the levels of HLA presentations, yet affected the rates of production of both the HLA peptidome and cellular proteome, mostly decreasing, but also increasing some of the synthesis rates of the HLA peptides and cellular proteins. Thus, we suggest that the degree of contribution of the proteasomal pathway to the production of the HLA-I peptidome should be re-evaluated in accordance with their effects on the entire HLA class-I peptidome, while taking into consideration the inhibitors'' effects on the synthesis (and degradation) rates of the source proteins of each of the studied HLA peptides.     

A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides

Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.Cross-linking/mass spectrometry extends the use of mass-spectrometry-based proteomics from identification (1, 2), quantification (3), and characterization of protein complexes (4) into resolving protein structures and protein–protein interactions (5–8). Chemical reagents (cross-linkers) covalently connect amino acid pairs that are within a cross-linker-specific distance range in the native three-dimensional structure of a protein or protein complex. A cross-linking/mass spectrometry experiment is typically conducted in four steps: (1) cross-linking of the target protein or complex, (2) protein digestion (usually with trypsin), (3) LC-MS analysis, and (4) database search. The digested peptide mixture consists of linear and cross-linked peptides, and the latter can be enriched by strong cation exchange (9) or size exclusion chromatography (10). Cross-linked peptides are of high value as they provide direct information on the structure and interactions of proteins.Cross-linked peptides fragment under collision-induced dissociation (CID) conditions primarily into b- and y-ions, as do their linear counterparts. An important difference regarding database searches between linear and cross-linked peptides stems from not knowing which peptides might be cross-linked. Therefore, one has to consider each single peptide and all pairwise combinations of peptides in the database. Having n peptides leads to (n2 + n)/2 possible pairwise combinations. This leads to two major challenges: With increasing size of the database, search time and the risk of identifying false positives increases. One way of circumventing these problems is to use MS2-cleavable cross-linkers (11, 12), at the cost of limited experimental design and choice of cross-linker.In a first database search approach (13), all pairwise combinations of peptides in a database were considered in a concatenated and linearized form. Thereby, all possible single bond fragments are considered in one of the two database entries per peptide pair, and the cross-link can be identified by a normal protein identification algorithm. Already, the second search approach split the peptides for the purpose of their identification (14). Linear fragments were used to retrieve candidate peptides from the database that are then matched based on the known mass of the cross-linked pair and scored as a pair against the spectrum. Isotope-labeled cross-linkers were used to sort the linear and cross-linked fragments apart. Many other search tools and approaches have been developed since (10, 15–19); see (20) for a more detailed list, at least some of which follow the general idea of an open modification search (21–24).As a general concept for open modification search of cross-linked peptides, cross-linked peptides represent two peptides, each with an unknown modification given by the mass of the other peptide and the cross-linker. One identifies both peptides individually and then matches them based on knowing the mass of cross-linked pair (14, 22, 24). Alternatively, one peptide is identified first and, using that peptide and the cross-linker as a modification mass, the second peptide is identified from the database (21, 23). An important element of the open modification search approach is that it essentially converts the quadratic search space of the cross-linked peptides into a linear search space of modified peptides. Still, many peptides and many modification positions have to be considered, especially when working with large databases or when using highly reactive cross-linkers with limited amino acid selectivity (25).We hypothesize that detailed knowledge of the fragmentation behavior of cross-linked peptides might reveal ways to improve the identification of cross-linked peptides. Detailed analyses of the fragmentation behavior of linear peptides exist (26–28), and the analysis of the fragmentation behavior of cross-linked peptides has guided the design of scores (24, 29). Further, cross-link-specific ions have been observed from higher energy collision dissociation (HCD) data (30). Isotope-labeled cross-linkers are used to distinguish cross-linked from linear fragments, generally in low-resolution MS2 of cross-linked peptides (14).We compared the mass spectrometric behavior of cross-linked peptides to that of linear peptides, using 910 high-resolution fragment spectra matched to unique cross-linked peptides from multiple different public datasets at 5% peptide-spectrum match (PSM)¹ false discovery rate (FDR). In addition, we repeated all experiments with a larger sample set that contains 8,301 spectra—also including data from ongoing studies from our lab (Supplemental material S9-S12). This paper presents the mass spectrometric signature of cross-linked peptides that we identified in our analysis and the resulting heuristics that are incorporated into an integrated strategy for the analysis and identification of cross-linked peptides. We present computational strategies that indicate the possibility of alleviating the need for mass-spectrometrically restricted cross-linker choice.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

Sampling From the Proteome to the Human Leukocyte Antigen-DR (HLA-DR) Ligandome Proceeds Via High Specificity

Comprehensive analysis of the complex nature of the Human Leukocyte Antigen (HLA) class II ligandome is of utmost importance to understand the basis for CD4⁺ T cell mediated immunity and tolerance. Here, we implemented important improvements in the analysis of the repertoire of HLA-DR-presented peptides, using hybrid mass spectrometry-based peptide fragmentation techniques on a ligandome sample isolated from matured human monocyte-derived dendritic cells (DC). The reported data set constitutes nearly 14 thousand unique high-confident peptides, i.e. the largest single inventory of human DC derived HLA-DR ligands to date. From a technical viewpoint the most prominent finding is that no single peptide fragmentation technique could elucidate the majority of HLA-DR ligands, because of the wide range of physical chemical properties displayed by the HLA-DR ligandome. Our in-depth profiling allowed us to reveal a strikingly poor correlation between the source proteins identified in the HLA class II ligandome and the DC cellular proteome. Important selective sieving from the sampled proteome to the ligandome was evidenced by specificity in the sequences of the core regions both at their N- and C- termini, hence not only reflecting binding motifs but also dominant protease activity associated to the endolysosomal compartments. Moreover, we demonstrate that the HLA-DR ligandome reflects a surface representation of cell-compartments specific for biological events linked to the maturation of monocytes into antigen presenting cells. Our results present new perspectives into the complex nature of the HLA class II system and will aid future immunological studies in characterizing the full breadth of potential CD4⁺ T cell epitopes relevant in health and disease.Human Leukocyte Antigen (HLA)¹ class II molecules on professional antigen presenting cells such as dendritic cells (DC) expose peptide fragments derived from exogenous and endogenous proteins to be screened by CD4⁺ T cells (1, 2). The activation and recruitment of CD4⁺ T cells recognizing disease-related peptide antigens is critical for the development of efficient antipathogen or antitumor immunity. Furthermore, the presentation of self-peptides and their interaction with CD4⁺ T cells is essential to maintain immunological tolerance and homeostasis (3). Knowledge of the nature of HLA class II-presented peptides on DC is of great importance to understand the rules of antigen processing and peptide binding motifs (4), whereas the identity of disease-related antigens may provide new knowledge on immunogenicity and leads for the development of vaccines and immunotherapy (5, 6).Mass spectrometry (MS) has proven effective for the analysis HLA class II-presented peptides (4, 7, 8). MS-based ligandome studies have demonstrated that HLA class II molecules predominantly present peptides derived from exogenous proteins that entered the cells by endocytosis and endogenous proteins that are associated with the endo-lysosomal compartments (4). Yet proteins residing in the cytosol, nucleus or mitochondria can also be presented by HLA class II molecules, primarily through autophagy (9–11). Multiple studies have mapped the HLA class II ligandome of antigen presenting cells in the context of infectious pathogens (12), autoimmune diseases (13–17) or cancer (14, 18, 19), or those that are essential for self-tolerance in the human thymus (3, 20). Notwithstanding these efforts, and certainly not in line with the extensive knowledge on the HLA class I ligandome (21), the nature of the HLA class II-presented peptide repertoire and particular its relationship to the cellular source proteome remains poorly understood.To advance our knowledge on the HLA-DR ligandome on activated DC without having to deal with limitations in cell yield from peripheral human blood (12, 21, 22) or tissue isolates (3), we explored the use of MUTZ-3 cells. This cell line has been used as a model of human monocyte-derived DCs. MUTZ-3 cells can be matured to act as antigen presenting cells and express then high levels of HLA class II molecules, and can be propagated in vitro to large cell densities (23–25). We also evaluated the performance of complementary and hybrid MS fragmentation techniques electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD) (26), and higher-energy collision dissociation (HCD) to sequence and identify the HLA class II ligandome. Together this workflow allowed for the identification of an unprecedented large set of about 14 thousand unique peptide sequences presented by DC derived HLA-DR molecules, providing an in-depth view of the complexity of the HLA class II ligandome, revealing underlying features of antigen processing and surface-presentation to CD4⁺ T cells.     

A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by Mass Spectrometry (MS)

Glycosylation is one of the most common and important protein modifications in biological systems. Many glycoproteins naturally occur at low abundances, which makes comprehensive analysis extremely difficult. Additionally, glycans are highly heterogeneous, which further complicates analysis in complex samples. Lectin enrichment has been commonly used, but each lectin is inherently specific to one or several carbohydrates, and thus no single or collection of lectin(s) can bind to all glycans. Here we have employed a boronic acid-based chemical method to universally enrich glycopeptides. The reaction between boronic acids and sugars has been extensively investigated, and it is well known that the interaction between boronic acid and diols is one of the strongest reversible covalent bond interactions in an aqueous environment. This strong covalent interaction provides a great opportunity to catch glycopeptides and glycoproteins by boronic acid, whereas the reversible property allows their release without side effects. More importantly, the boronic acid-diol recognition is universal, which provides great capability and potential for comprehensively mapping glycosylation sites in complex biological samples. By combining boronic acid enrichment with PNGase F treatment in heavy-oxygen water and MS, we have identified 816 N-glycosylation sites in 332 yeast proteins, among which 675 sites were well-localized with greater than 99% confidence. The results demonstrated that the boronic acid-based chemical method can effectively enrich glycopeptides for comprehensive analysis of protein glycosylation. A general trend seen within the large data set was that there were fewer glycosylation sites toward the C termini of proteins. Of the 332 glycoproteins identified in yeast, 194 were membrane proteins. Many proteins get glycosylated in the high-mannose N-glycan biosynthetic and GPI anchor biosynthetic pathways. Compared with lectin enrichment, the current method is more cost-efficient, generic, and effective. This method can be extensively applied to different complex samples for the comprehensive analysis of protein glycosylation.Glycosylation is an extremely important protein modification that frequently regulates protein folding, trafficking, and stability. It is also involved in a wide range of cellular events (1) such as immune response (2, 3), cell proliferation (4), cell-cell interactions (5), and signal transduction (6). Aberrant protein glycosylation is believed to have a direct correlation with the development of several diseases, including diabetes, infectious diseases, and cancer (7–11). Secretory proteins frequently get glycosylated, including those in body fluids such as blood, saliva, and urine (12, 13). Samples containing these proteins can be easily obtained and used for diagnostic and therapeutic purposes. Several glycoproteins have previously been identified as biomarkers, including Her2/Neu in breast cancer (14), prostate-specific antigen (PSA) in prostate cancer (15), and CA125 in ovarian cancer (16, 17), which highlights the clinical importance of identifying glycoproteins as indicators or biomarkers of diseases. Therefore, effective methods for systematic analysis of protein glycosylation are essential to understand the mechanisms of glycobiology, identify drug targets and discover biomarkers.Approximately half of mammalian cell proteins are estimated to be glycosylated at any given time (18). There have been many reports regarding identification of protein glycosylation sites and elucidation of glycan structures (19–30). Glycan structure analysis can lead to potential therapeutic and diagnostic applications (31, 32), but it is also critical to identify which proteins are glycosylated as well as the sites at which the modification occurs. Despite progress in recent years, the large-scale analysis of protein glycosylation sites using MS-based proteomics methods is still a challenge. Without an effective enrichment method, the low abundance of glycoproteins prohibits the identification of the majority of sites using the popular intensity-dependent MS sequence method.About a decade ago, a very beautiful and elegant method based on hydrazide chemistry was developed to enrich glycopeptides. Hydrazide conjugated beads reacted with aldehydes formed from the oxidation of cis-diols in glycans (33). This method has been extensively applied to many different types of biological samples (34–41). Besides the hydrazide-based enrichment method, lectins have also been frequently used to enrich glycopeptides or glycoproteins before MS analysis (28, 29, 42–46). However, there are many different types of lectins, and each is specific to certain glycans (47, 48). Therefore, no combination of lectins can bind to all glycosylated peptides or proteins, which prevents comprehensive analysis of protein glycosylation. Because of the complexity of biological samples, effective enrichment methods are critical for the comprehensive analysis of protein glycosylation before MS analysis.One common feature of all glycoproteins and glycopeptides is that they contain multiple hydroxyl groups in their glycans. From a chemistry point of view, this can be exploited to effectively enrich them. Ideally, chemical enrichment probes must have both strong and specific interactions with multiple hydroxyl groups. The reaction between boronic acids and 1,2- or 1,3-cis-diols in sugars has been extensively studied (49–52) and applied for the small-scale analysis of glycoproteins (53–55). Furthermore, boronate affinity chromatography has been employed for the analysis of nonenzymatically glycated peptides (56, 57). Boronic acid-based chemical enrichment methods are expected to have great potential for global analysis of glycopeptides when combined with modern MS-based proteomics techniques. However, the method has not yet been used for the comprehensive analysis of protein N-glycosylation in complex biological samples (58).Yeast is an excellent model biological system that has been extensively used in a wide range of experiments. Last year, two papers reported the large-scale analysis of protein N-glycosylation in yeast (59, 60). In one study, a new MS-based method was developed based on N-glycopeptide mass envelopes with a pattern via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides with the recoded envelopes were specifically targeted for fragmentation, facilitating high confidence site mapping (59). Using this method, 133 N-glycosylation sites were confidently identified in 58 yeast proteins. When combined with an effective enrichment method, this MS-based analysis will provide a more complete coverage of the N-glycoproteome. The other work combined lectin enrichment with digestion by two enzymes (Glu_c and trypsin) to increase the peptide coverage, and 516 well-localized N-glycosylation sites were identified in 214 yeast proteins by MS (60).Here we have comprehensively identified protein N-glycosylation sites in yeast by combining a boronic acid-based chemical enrichment method with MS-based proteomics techniques. Magnetic beads conjugated with boronic acid were systematically optimized to selectively enrich glycosylated peptides from yeast whole cell lysates. The enriched peptides were subsequently treated with Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)¹ in heavy-oxygen water. Finally, peptides were analyzed by an on-line LC-MS system. Over 800 protein N-glycosylation sites were identified in the yeast proteome, which clearly demonstrates that the boronic acid-based chemical method is an effective enrichment method for large-scale analysis of protein glycosylation by MS.     

JUMP: A Tag-based Database Search Tool for Peptide Identification with High Sensitivity and Accuracy

Database search programs are essential tools for identifying peptides via mass spectrometry (MS) in shotgun proteomics. Simultaneously achieving high sensitivity and high specificity during a database search is crucial for improving proteome coverage. Here we present JUMP, a new hybrid database search program that generates amino acid tags and ranks peptide spectrum matches (PSMs) by an integrated score from the tags and pattern matching. In a typical run of liquid chromatography coupled with high-resolution tandem MS, more than 95% of MS/MS spectra can generate at least one tag, whereas the remaining spectra are usually too poor to derive genuine PSMs. To enhance search sensitivity, the JUMP program enables the use of tags as short as one amino acid. Using a target-decoy strategy, we compared JUMP with other programs (e.g. SEQUEST, Mascot, PEAKS DB, and InsPecT) in the analysis of multiple datasets and found that JUMP outperformed these preexisting programs. JUMP also permitted the analysis of multiple co-fragmented peptides from “mixture spectra” to further increase PSMs. In addition, JUMP-derived tags allowed partial de novo sequencing and facilitated the unambiguous assignment of modified residues. In summary, JUMP is an effective database search algorithm complementary to current search programs.Peptide identification by tandem mass spectra is a critical step in mass spectrometry (MS)-based¹ proteomics (1). Numerous computational algorithms and software tools have been developed for this purpose (2–6). These algorithms can be classified into three categories: (i) pattern-based database search, (ii) de novo sequencing, and (iii) hybrid search that combines database search and de novo sequencing. With the continuous development of high-performance liquid chromatography and high-resolution mass spectrometers, it is now possible to analyze almost all protein components in mammalian cells (7). In contrast to rapid data collection, it remains a challenge to extract accurate information from the raw data to identify peptides with low false positive rates (specificity) and minimal false negatives (sensitivity) (8).Database search methods usually assign peptide sequences by comparing MS/MS spectra to theoretical peptide spectra predicted from a protein database, as exemplified in SEQUEST (9), Mascot (10), OMSSA (11), X!Tandem (12), Spectrum Mill (13), ProteinProspector (14), MyriMatch (15), Crux (16), MS-GFDB (17), Andromeda (18), BaMS² (19), and Morpheus (20). Some other programs, such as SpectraST (21) and Pepitome (22), utilize a spectral library composed of experimentally identified and validated MS/MS spectra. These methods use a variety of scoring algorithms to rank potential peptide spectrum matches (PSMs) and select the top hit as a putative PSM. However, not all PSMs are correctly assigned. For example, false peptides may be assigned to MS/MS spectra with numerous noisy peaks and poor fragmentation patterns. If the samples contain unknown protein modifications, mutations, and contaminants, the related MS/MS spectra also result in false positives, as their corresponding peptides are not in the database. Other false positives may be generated simply by random matches. Therefore, it is of importance to remove these false PSMs to improve dataset quality. One common approach is to filter putative PSMs to achieve a final list with a predefined false discovery rate (FDR) via a target-decoy strategy, in which decoy proteins are merged with target proteins in the same database for estimating false PSMs (23–26). However, the true and false PSMs are not always distinguishable based on matching scores. It is a problem to set up an appropriate score threshold to achieve maximal sensitivity and high specificity (13, 27, 28).De novo methods, including Lutefisk (29), PEAKS (30), NovoHMM (31), PepNovo (32), pNovo (33), Vonovo (34), and UniNovo (35), identify peptide sequences directly from MS/MS spectra. These methods can be used to derive novel peptides and post-translational modifications without a database, which is useful, especially when the related genome is not sequenced. High-resolution MS/MS spectra greatly facilitate the generation of peptide sequences in these de novo methods. However, because MS/MS fragmentation cannot always produce all predicted product ions, only a portion of collected MS/MS spectra have sufficient quality to extract partial or full peptide sequences, leading to lower sensitivity than achieved with the database search methods.To improve the sensitivity of the de novo methods, a hybrid approach has been proposed to integrate peptide sequence tags into PSM scoring during database searches (36). Numerous software packages have been developed, such as GutenTag (37), InsPecT (38), Byonic (39), DirecTag (40), and PEAKS DB (41). These methods use peptide tag sequences to filter a protein database, followed by error-tolerant database searching. One restriction in most of these algorithms is the requirement of a minimum tag length of three amino acids for matching protein sequences in the database. This restriction reduces the sensitivity of the database search, because it filters out some high-quality spectra in which consecutive tags cannot be generated.In this paper, we describe JUMP, a novel tag-based hybrid algorithm for peptide identification. The program is optimized to balance sensitivity and specificity during tag derivation and MS/MS pattern matching. JUMP can use all potential sequence tags, including tags consisting of only one amino acid. When we compared its performance to that of two widely used search algorithms, SEQUEST and Mascot, JUMP identified ∼30% more PSMs at the same FDR threshold. In addition, the program provides two additional features: (i) using tag sequences to improve modification site assignment, and (ii) analyzing co-fragmented peptides from mixture MS/MS spectra.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P_s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS³ in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1–3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ297631","term_id":"9931190","term_text":"AJ297631"}}AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6–8). Investigations showed that treatment of sperm with α-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9–11), sperm motility (9–13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15–17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18, 19), providing the specificity required for a potential contraceptive. Questions surrounding these particular compounds were raised when a number of side effects were evident from in vivo trials (7, 20–22); however, the design of small molecule inhibitors of GAPDS may provide a viable alternative. Its potential as a contraceptive target was supported by data from mice where GAPDS^−/− males (23) were infertile because of defects in sperm motility.Glyceraldehyde-3-phosphate dehydrogenases are tetrameric enzymes that catalyze the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (Glc-3-P) into 1,3-diphosphoglycerate in the presence of an NAD cofactor via a two-step chemical mechanism (24). The first models of substrate binding were proposed on the basis of crystal structures of the holoenzyme from lobster (25) and Bacillus stearothermophilus (26), and Moras and co-workers (25) identified two anion-binding sites postulated to correspond to those binding the C-3 phosphate group of d-Glc-3-P (P_s site) and the inorganic phosphate ion (P_i site).Structure-based design of small molecules to inhibit GAPDH is not unprecedented. GAPDH has been targeted from protozoan parasites (27–30), as the bloodstream forms rely solely on glycolysis for energy production (31, 32). A number of mammalian GAPDH structures have also been solved, including rabbit muscle (33, 34), human liver (35), and human placenta (36); however, no structures are available for sperm-specific isoforms of this enzyme.Active heterotetramers of GAPDH between different species have been reported and biochemically characterized previously, both in ratios of 2:2 and 3:1 (37–40). In this study we have successfully obtained crystals of rat recombinant GAPDS as a heterotetramer with Escherichia coli GAPDH in a 1:3 ratio. To understand the basis of inhibition of the sperm isoform by substrate analogue 3-chlorolactaldehyde, a metabolite of α-chlorohydrin, a structure was also determined in the presence of the substrate glyceraldehyde 3-phosphate. The sperm-specific structure was compared with the human placental GAPDH structure (PDB entry 1U8F; Ref. 36) to identify differences that may provide a target for the design of inhibitors specific to the GAPDS protein. The unique structural features identified offer potential candidates for further investigation as inhibitor targets.     

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS³ analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS³) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.Proteins form stable and dynamic multisubunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multisubunit protein complexes has been difficult because of a lack of technologies that can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and x-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent, whereas x-ray crystallography is often limited by the crystallization process.In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions (1–3). Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues (4–9). In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low resolution structural information about the constraints within a molecule (2, 3, 10) or protein complex (11–13). The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analyses for the elucidation of three-dimensional protein structures offers distinct advantages over traditional methods because of its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing the structural topology of proteins and/or protein complexes. Although this approach has been successful, effective detection and accurate identification of cross-linked peptides as well as unambiguous assignment of cross-linked sites remain extremely challenging due to their low abundance and complicated fragmentation behavior in MS analysis (2, 3, 10, 14). Therefore, new reagents and methods are urgently needed to allow unambiguous identification of cross-linked products and to improve the speed and accuracy of data analysis to facilitate its application in structural elucidation of large protein complexes.A number of approaches have been developed to facilitate MS detection of low abundance cross-linked peptides from complex mixtures. These include selective enrichment using affinity purification with biotinylated cross-linkers (15–17) and click chemistry with alkyne-tagged (18) or azide-tagged (19, 20) cross-linkers. In addition, Staudinger ligation has recently been shown to be effective for selective enrichment of azide-tagged cross-linked peptides (21). Apart from enrichment, detection of cross-linked peptides can be achieved by isotope-labeled (22–24), fluorescently labeled (25), and mass tag-labeled cross-linking reagents (16, 26). These methods can identify cross-linked peptides with MS analysis, but interpretation of the data generated from interlinked peptides (two peptides connected with the cross-link) by automated database searching remains difficult. Several bioinformatics tools have thus been developed to interpret MS/MS data and determine interlinked peptide sequences from complex mixtures (12, 14, 27–32). Although promising, further developments are still needed to make such data analyses as robust and reliable as analyzing MS/MS data of single peptide sequences using existing database searching tools (e.g. Protein Prospector, Mascot, or SEQUEST).Various types of cleavable cross-linkers with distinct chemical properties have been developed to facilitate MS identification and characterization of cross-linked peptides. These include UV photocleavable (33), chemical cleavable (19), isotopically coded cleavable (24), and MS-cleavable reagents (16, 26, 34–38). MS-cleavable cross-linkers have received considerable attention because the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis. Gas-phase cleavage sites result in the detection of a “reporter” ion (26), single peptide chain fragment ions (35–38), or both reporter and fragment ions (16, 34). In each case, further structural characterization of the peptide product ions generated during the cleavage reaction can be accomplished by subsequent MSⁿ1 analysis. Among these linkers, the “fixed charge” sulfonium ion-containing cross-linker developed by Lu et al. (37) appears to be the most attractive as it allows specific and selective fragmentation of cross-linked peptides regardless of their charge and amino acid composition based on their studies with model peptides.Despite the availability of multiple types of cleavable cross-linkers, most of the applications have been limited to the study of model peptides and single proteins. Additionally, complicated synthesis and fragmentation patterns have impeded most of the known MS-cleavable cross-linkers from wide adaptation by the community. Here we describe the design and characterization of a novel and simple MS-cleavable cross-linker, DSSO, and its application to model peptides and proteins and the yeast 20 S proteasome complex. In combination with new software developed for data integration, we were able to identify DSSO-cross-linked peptides from complex peptide mixtures with speed and accuracy. Given its effectiveness and simplicity, we anticipate a broader application of this MS-cleavable cross-linker in the study of structural topology of other protein complexes using cross-linking and mass spectrometry.