Mass Spectrometric Peptide Research Articles

Proteomic and Functional Consequences of Hexokinase Deficiency in Glucose-repressible Kluyveromyces lactis

The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K. lactis proteins that were solubilized from glucose-grown KlHxk1 wild-type and mutant cells. Mass spectrometric peptide analysis identified 45 individual hexokinase-dependent proteins related to carbohydrate, short-chain fatty acid and tricarboxylic acid metabolism as well as to amino acid and protein turnover, but also to general stress response and chromatin remodeling, which occurred as a consequence of KlHxk1 deficiency at a minimum 3-fold enhanced or reduced level in the mutant proteome. In addition, three proteins exhibiting homology to 2-methylcitrate cycle enzymes of S. cerevisiae were detected at increased concentrations, suggesting a stimulation of pyruvate formation from amino acids and/or fatty acids. Experimental validation of the difference gel electrophoresis approach by post-lysis dimethyl labeling largely confirmed the abundance changes detected in the mutant proteome via the former method. Taking into consideration the high proportion of identified hexokinase-dependent proteins exhibiting increased proteomic levels, KlHxk1 is likely to have a repressive function in a multitude of metabolic pathways. The proteomic alterations detected in the mutant classify KlHxk1 as a multifunctional enzyme and support the view of evolutionary conservation of dual-role hexokinases even in organisms that are less specialized than S. cerevisiae in terms of glucose utilization.

Induction, characterization, and heterologous expression of a carotenoid degrading versatile peroxidase from Pleurotus sapidus

Abstract A β-carotene degrading versatile peroxidase (VP) was successfully induced in submerged cultures of the white-rot basidiomycete Pleurotus sapidus by use of residues of a biogas plant as a carbon and nitrogen source. After three chromatographic steps, the VP was isolated with an overall yield of 12% and a purification factor of 130. The purified enzyme showed a molecular mass of 38 kDa and an isoelectric point of 3.6. Highest affinity to β-carotene ( K m = 50 ± 5 μM) was observed at 30 °C and pH 4.5. The purified VP was capable of degrading suspended lignin organosolv particles. N-terminal and internal peptide sequences were obtained from Edman degradation and mass spectrometric peptide sequencing. The VP encoding cDNA was identified by colony hybridization and amplified by PCR. Bioinformatic analyses revealed an open reading frame of 1083 bp and similarities of 90% to VPs from P. eryngii . The recombinant VP was produced successfully with an activity of 450 ± 20 mU mg −1 in cultures of H. polymorpha .

Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation

The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other (13)C or (15)N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce (34)S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response.

Mass spectrometric peptide analysis of 2DE‐separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation

Asparagine deamidation is a common nonenzymatic post-translational modification comprising the conversion of asparaginyl residues to aspartyl and isoaspartyl residues, respectively. As a result an additional negative charge is introduced that can affect the tertiary structure as well as the biological activity of a protein. Since deamidation reduces the protein's pI value, differentially deamidated forms of a protein can be separated in 2D gels. We have analyzed a dataset of 430 protein spots from 2D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a Coomassie-stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin-2, V-type proton ATPase subunit B, and protein disulfide-isomerase A3 were also found in 2D gels of proteins extracted from rat hippocampus. All identified deamidation sites contained a glycine residue on the carboxyl side of the asparaginyl residue. Strikingly, a second glycine residue at the +3 position was found in the majority of the deamidated peptides. We propose that the NGxG motif confers exceptional susceptibility to in vivo asparagine deamidation.

Structural consequences of dry heating on alpha-lactalbumin and beta-lactoglobulin at pH 6.5

In the present work, we investigated the structural modifications occurring during the dry heating of model whey proteins, β-lactoglobulin and α-lactalbumin. Samples were adjusted to pH 6.5, water activity aw=0.23 and dry heated at 100°C for up to 24h, and the structural modifications followed by gel permeation chromatography, reverse phase-HPLC, SDS PAGE and mass spectrometry (LC–MS/MS). The dry heating treatment traps a fraction of the proteins into covalently linked soluble aggregates. Moreover, a high proportion of non-aggregated α-lactalbumin (about 73%) was converted into non-native forms. The characteristic of those non-native species was the loss of one or two water molecules per α-lactalbumin molecules. Using tandem mass spectrometric peptide mapping, these chemical modifications were found to be attributed to (i) the formation of a pyroglutamic acid from the N-terminal glutamic acid and (ii) the formation of an internal cyclic imide at position Asp64. The non-native species were not favored in the case of β-lactoglobulin as they represented less than 18% of non-aggregated proteins.

Native chromatographic sample preparation of serum, plasma and cerebrospinal fluid does not comprise a risk for proteolytic biomarker loss

We recently developed a native multidimensional chromatographic method for serum and plasma fractionation for proteomic biomarker search. This method has several advantages:parallelization and automation, high reproducibility and proteome coverage, flexible dynamic range with respect to molecular weight and sample amount, optional enzymatic and immunological analytics additional to mass spectrometry, retaining metabolites, and information on complex formation, modification, and fragmentation of constituents. Nevertheless, native conditions have the probable risk of proteome alteration and biomarker loss by intrinsic proteinases. Hence, we tried to quantify here intrinsic proteolytic activity in native samples and fractions from serum, plasma and cerebrospinal fluid, as well as the effectiveness of intrinsic anti-proteinases during sample handling and preparation under our fractionation conditions. Therefore, we used several quantitative measures: (1) total proportion of intrinsic protein and peptide fractions, (2) azocasein hydrolysis and (3) mass spectrometric protein coverage and peptide numbers. To 1: In all non-fractionated specimens, neither decrease of protein concentration or molecular weight nor increase of peptide concentration was found after variable clotting or pre-incubation time. To 2: No azocasein hydrolysis was seen in these samples when prepared within a few hours at room temperature. Trypsin, when added in concentrations not higher than 0.85 μg/mL (0.04 μM), even was completely inhibited. Moreover, in native 1-D fractions no proteinase activity could be observed. To 3: Mass spectrometry confirmed that neither protein coverage nor peptide numbers differ significantly in 1-D or 2-D fractions after variable incubation time. These results suggest that intrinsic, native proteinase inhibitors potentially protect the proteomes considered, enabling “top-down” proteomic approaches under native conditions with serum, plasma and cerebrospinal fluid.

Characterization of a high‐affinity human antibody with a disulfide bridge in the third complementarity‐determining region of the heavy chain

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.

Combined Electron Transfer Dissociation–Collision-Induced Dissociation Fragmentation in the Mass Spectrometric Distinction of Leucine, Isoleucine, and Hydroxyproline Residues in Peptide Natural Products

Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

Matrix-assisted laser desorption/ionization mass spectrometry peptide sequencing utilizing selective N-terminal bromoacetylation

In tandem mass spectrometric peptide sequencing, simplifying the mass spectrum is often desirable. The b-series ions were distinguished from the y-series ions in the MALDI TOF-TOF spectra by incorporating a bromine-tag to the N-terminal amino group through rapid and selective acetylation using bromoacetic anhydride without blocking the lysine and tyrosine residues. The 51:49 ratio of Br-79 and Br-81 isotopes facilitated identification of ions carrying the tag. With the Br-tag in the b-series ions, N-terminal sequencing of tryptic peptides from hemoglobin as well as model peptides was straightforward. When the b-ions were low in intensity, ions without the Br-tag were identified as y-ions and used for sequencing.

Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity

OBJECTIVETo study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity.RESEARCH DESIGN AND METHODSHuman LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping.RESULTSMGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding.CONCLUSIONSMG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria

Phototrophic epilithic biofilms harbour a distinct assemblage of heterotrophic bacteria, cyanobacteria and photoautotrophic algae. Secretion of extracellular polymeric substances (EPS) by these organisms and the physicochemical properties of the EPS are important factors for the development of the biofilms. We have isolated representative diatom and bacteria strains from epilithic biofilms of Lake Constance. By pairwise co-cultivating these strains we found that diatom growth and EPS secretion by diatoms may depend on the presence of individual bacteria. Similar results were obtained after addition of spent bacterial medium to diatom cultures, suggesting that soluble substances from bacteria have an impact on diatom physiology. While searching for putative bacterial signal substances, we found that concentrations of various dissolved free amino acids (DFAA) within the diatom cultures changed drastically during co-cultivation with bacteria. Further, the secretion of extracellular carbohydrates and proteins can be influenced by bacteria or their extracellular substances. We have performed mass spectrometric peptide mapping to identify proteins which are secreted when co-cultivating the diatom Phaeodactylum tricornutum Bohlin and Escherichia coli. The identified proteins are possibly involved in signalling, extracellular carbohydrate modification and uptake, protein and amino acid modification, and cell/cell aggregation of diatom and bacteria strains. Our data indicate that diatom-bacteria biofilms might be regulated by a complex network of chemical factors involving EPS, amino acid monomers and other substances. Thus interactions with bacteria can be considered as one of the main factors driving biofilm formation by benthic diatoms.

Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi

The use of MALDI-TOF and other types of mass spectrometry for the identification and investigation of extracellular enzymes (carbohydrases, proteinases, esterases, etc.) produced by filamentous fungi belonging to the genera Aspergillus, Trichoderma, Penicilium, Chrysosporium, etc. is discussed. The method of mass spectrometric peptide fingerprinting combined with the use of Internet software for on line data analysis (MASCOT, Aldente, FindPept, FindMod, GlycoMod) allows the fast and reliable identification of both individual enzymes and the components of crude multienzyme preparations without their fractionation. The method was also applied to the discrimination of the fungal genes encoding enzymes with similar substrate specificity. Other enzyme-based applications of mass spectrometry, such as the revelation of the presence or absence of structural domains in the molecules of carbohydrases, the detection of posttranslational and artificial modifications in the enzymes, and the use of tandem mass spectrometry for de novo peptide sequencing are described.

Enzastaurin Treatment Affects Multiple Regulatory Pathways at Transcriptome and Cellular Proteome Level of Mantle Cell Lymphoma

AbstractAbstract 2893 Background:The protein kinase C beta inhibitor enzastaurin is one of the promising molecular targeted approaches currently investigated in mantle cell lymphoma (MCL), a disease still characterized by a dismal long term prognosis. Methods:Four well characterized MCL cell lines (Granta 519, HBL-2, Jeko-1 and Rec-1) as well as three patient samples were exposed to enzastaurin at a previously defined dose (10 μM). Cell viability as well as cell cycle activity were analyzed by tryphan blue exclusion test and flow cytometry, respectively, after 24 and 48 hours. To dissect the regulatory processes targeted by enzastaurin, the panel of MCL cell lines was screened on both protein and RNA expression levels (2D-gel electrophoresis and mass spectrometric peptide fingerprint analysis and Affymetrix microarray) after 4h enzastaurin treatment. Results:Enzastaurin in vitro resulted in a reduced viability and cell proliferation by 15–20% after 24h in cell lines and 9–20% in primary patient samples after 48h. This effect was related to a G2/M block of cell cycle and induction of apoptosis. Based on the proteome and transcriptome analysis of early alterations, only HSPD1 was affected on both regulation levels. Nonetheless, combined analysis of alterations on both, protein and RNA expression levels, resulted in identification of common signal pathways characterizing a more comprehensive network of affected molecular interactions mapping to distinct canonical pathways and defined cellular functions. Indicated canonical pathways included ‘calcium signalling’, (CAMKK2, HDAC5, HDAC9, TP63) ‘calcium induced T-lymphocyte apoptosis’ (MEF2D, NR4A1, PRKCG, TRA@), ‘NFkB signalling’ (KRAS, MAP3K8, TNFAIP3, TNFRS17) and ‘molecular mechanisms of cancer’ (APAF1, CDKN2D, FOS, PAK6), whereas the top ranking cellular functions were ‘cellular growth and proliferation’ (CCNG2, EIF4E, PDIA3, TOP1, TPM1,), ‘cell death’ (BCL6, EEF1D, PAK6, RAD50), 'cell cycle'(AKAP9, BMF, CUL5, GADD45B, PDIA3), 'cellular development' (APAF1, GAS7, ID1, PAX8) and ‘gene expression’ (ABCG1, HOXB4, LMO4, PIM1). Alterations of these pathways were confirmed by Western Blot analysis of selected candidate proteins marker proteins of the regulated pathways. Conclusion:In summary, the combined approach of RNA and protein analysis revealed the targeted signal pathways after Enzastaurin exposure. These data will allow a more rationally designed combination of biologicals to finally improve the clinical outcome of MCL. Disclosures:Dreyling:Eli Lilly: Support of in vitro studies of Enzostaurin in MCL.

MassSieve: Panning MS/MS peptide data for proteins

We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments.

Surface‐activated chemical ionization time‐of‐flight mass spectrometry and labeling‐free approach: two powerful tools for the analysis of complex plant functional proteome profiles

Surface-activated chemical ionization (SACI) has been widely used in recent years to analyze a range of different compounds (e.g., peptides, street drugs, amino acids). The main benefits of this technology are its high sensitivity and its effectiveness under different chromatographic conditions. Here, we used SACI in conjunction with a highly selective quadrupole time-of-flight mass analyzer to characterize a complex proteome pattern after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The data obtained were compared with those obtained using the micro-electrospray ionization (ESI) approach, which showed that using SACI strongly increased the number of detectable proteins. The higher sensitivity is mainly due to the ability of SACI to selectively produce singly charged species of high intensity under full-scan conditions and doubly charged species for tandem mass spectrometric (MS/MS) peptide characterization by simply changing the ionization conditions during data acquisition.

Regulation of gonadotropin-releasing hormone-1 gene transcription by members of the purine-rich element-binding protein family

Gonadotropin-releasing hormone-1 (GnRH1) controls reproduction by stimulating the release of gonadotropins from the pituitary. To characterize regulatory factors governing GnRH1 gene expression, we employed biochemical and bioinformatics techniques to identify novel GnRH1 promoter-binding proteins from the brain of the cichlid fish, Astatotilapia burtoni (A. burtoni). Using an in vitro DNA-binding assay followed by mass spectrometric peptide mapping, we identified two members of the purine-rich element-binding (Pur) protein family, Puralpha and Purbeta, as candidates for GnRH1 promoter binding and regulation. We found that transcripts for both Puralpha and Purbeta colocalize in GnRH1-expressing neurons in the preoptic area of the hypothalamus in A. burtoni brain. Furthermore, we confirmed in vivo binding of endogenous Puralpha and Purbeta to the upstream region of the GnRH1 gene in A. burtoni brain and mouse neuronal GT1-7 cells. Consistent with the relative promoter occupancy exhibited by endogenous Pur proteins, overexpression of Purbeta, but not Puralpha, significantly downregulated GnRH1 mRNA levels in transiently transfected GT1-7 cells, suggesting that Purbeta acts as a repressor of GnRH1 gene transcription.

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: a new tool in diagnostic investigation of nail disorders?

The incidence and prevalence of onychomycosis are rising worldwide. Common diagnostic techniques often lack sensitivity or specificity. Differentiation between non-infectious nail disorders is frequently not possible. The aim of this study was to establish a better diagnostic routine procedure based on modern mass spectrometric peptide analysis techniques. One hundred and fifty-five nail samples from 145 patients with clinically suspected onychomycosis (n = 96, 62%) and without onychomycosis [e.g. nail psoriasis or nail dystrophy resulting from eczema (n = 59, 38%)] were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) peptide mass fingerprinting in comparison with standard techniques. We demonstrated that MALDI-TOF MS represents a precise, robust and fast tool in diagnostic investigation of nail disorders, which is superior to common standard methods.

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K m,app = 0.16 ± 0.02 mg/mL and k cat,app = 79 ± 10 s −1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k cat,app/ K m,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with K d of 27.2, 0.70, and 34.7 μM, respectively.

A Carboxy-Terminal Affinity Tag for the Purification and Mass Spectrometric Characterization of Integral Membrane Proteins

G-protein-coupled receptors (GPCRs) and other structurally and functionally related membrane proteins represent particularly attractive targets for drug discovery. Integral membrane proteins are often difficult to purify from native contexts, and lack of sufficient quantities hampers subsequent structural and functional proteomic studies. We describe here an optimized enrichment strategy involving a membrane protein-compatible 1D4 affinity tag that is derived from the carboxy-terminal nine amino residues of bovine rhodopsin, and its corresponding tag-specific, high-affinity monoclonal antibody. When two GPCRs as well as two related ATP binding cassette (ABC) transporters are expressed in their functional forms in human cell lines, we have shown that a single detergent and wash condition can be employed for the purification of all said membrane proteins. Subsequent in-gel digestion with trypsin and mass spectrometric peptide analysis resulted in high sequence coverage for the ABC transporters ABCA1-1D4 and ABCA4-1D4. In contrast, digestion by various enzymatic combinations was necessary to obtain the best sequence coverage for affinity-enriched GPCRs CXCR4-1D4 and CCR5-1D4 as compared against other entries in an annotated spectrum library. Furthermore, specific enzyme combinations were necessary to produce suitable peptides for deducing N-glycosylation sites on CXCR4. Our results demonstrate that the 1D4-tag enrichment strategy is a versatile tool for the characterization of integral membrane proteins that can be employed for functional proteomic studies.

Identification of naturally processed ligands in the C57BL/6 mouse using large-scale mass spectrometric peptide sequencing and bioinformatics prediction

Most major histocompatibility complex (MHC) class I-peptide-binding motifs are currently defined on the basis of quantitative in vitro MHC-peptide-binding assays. This information is used to develop bioinformatics-based tools to predict the binding of peptides to MHC class I molecules. To date few studies have analyzed the performance of these bioinformatics tools to predict the binding of peptides determined by sequencing of naturally processed peptides eluted directly from MHC class I molecules. In this study, we performed large-scale sequencing of endogenous peptides eluted from H2K(b) and H2D(b) molecules expressed in spleens of C57BL/6 mice. Using sequence data from 281 peptides, we identified novel preferred anchor residues located in H2K(b) and H2D(b)-associated peptides that refine our knowledge of these H2 class I peptide-binding motifs. The analysis comparing the performance of three bioinformatics methods to predict the binding of these peptides, including artificial neural network, stabilized matrix method, and average relative binding, revealed that 61% to 94% of peptides eluted from H2K(b) and H2D(b) molecules were correctly classified as binders by the three algorithms. These results suggest that bioinformatics tools are reliable and efficient methods for binding prediction of naturally processed MHC class I ligands.