Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A comparison of MS/MS‐based,stable‐isotope‐labeled,quantitation performance on ESI‐quadrupole TOF and MALDI‐TOF/TOF mass spectrometers

The peptide‐based quantitation accuracy and precision of LC‐ESI (QSTAR Elite) and LC‐MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ‐labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC‐MALDI spectra. The average protein sequence coverages for LC‐ESI and LC‐MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ‐based expression ratios determined by ProteinPilot from the 57 467 ESI‐MS/MS and 26 085 MALDI‐MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7–6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC‐ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC‐MALDI iTRAQ ratios were rejected. Re‐analysis of an archived LC‐MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS‐based peptide quantitation performance of offline LC‐MALDI was comparable with on‐line LC‐ESI, which required threefold less time. However, offline LC‐MALDI allows the re‐analysis of archived HPLC‐separated samples.     

Authentication of the proteins expression profiles (PEPs) identification methodology in a bloom of Karenia digitata,the most damaging harmful algal bloom causative agent in the history of Hong Kong

Identification of harmful algal bloom (HAB) causative agents makes use either of morphology-based techniques or genetic tools. These techniques are often time-consuming, labor intensive, and/or based on subjective judgment. Recently, matching with protein/peptide expression profiles (PEPs) obtained with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF–MS) has emerged as a new technique for species identification of various microorganisms. We were the first group to adopt this rapid and simple methodology for the identification of dinoflagellates (Lee et al., 2008). In the present study, we evaluated the use of PEPs from MALDI–TOF–MS for species identification of field samples obtained from a local red-tide caused by Karenia digitata. PEPs obtained from direct MALDI–TOF–MS analysis of field samples were compared to that of established monospecific reference culture. Several species-specific peaks of K. digitata were found in the reference monoculture and most of these observed peaks could be matched to the PEPs of the field samples. Matched species-specific peaks observed from the PEPs of field samples would allow rapid identification of the causative agents in corresponding and future HABs. Furthermore, as K. digitata is the most damaging dinoflagellate in Hong Kong's history, we were interested in obtaining its SSU, partial LSU rDNA and ITS sequences for future studies. In the present study, besides reporting on the ITS and rDNA sequences of K. digitata, phylogenetic tree analysis was also performed. The results showed that K. digitata fell within the Karlodinium clade and had a closer relationship with the Karlodinium species than the Karenia species.     

Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years. Nonetheless, a need still exists for new digestion strategies that meet the demands of proteomics for high-throughput and rapid detection and identification of proteins. We performed an evaluation of direct tryptic digestion of proteins on a MALDI target plate and the potential for integrating RP HPLC separation of protein with on-target tryptic digestion in order to achieve a rapid and effective identification of proteins in complex biological samples. To this end, we used a Tempo HPLC/MALDI target plate deposition hybrid instrument (ABI). The technique was evaluated using a number of soluble and membrane proteins and an MRC5 cell lysate. We demonstrated that direct deposition of proteins on a MALDI target plate after reverse-phase HPLC separation and subsequent tryptic digestion of the proteins on the target followed by MALDI TOF/TOF analysis provided substantial data (intact protein mass, peptide mass and peptide fragment mass) that allowed a rapid and unambiguous identification of proteins. The rapid protein separation and direct deposition of fractions on a MALDI target plate provided by the RP HPLC combined with off-line interfacing with the MALDI MS is a unique platform for rapid protein identification with improved sequence coverage. This simple and robust approach significantly reduces the sample handling and potential loss in large-scale proteomics experiments. This approach allows combination of peptide mass fingerprinting (PMF), MS/MS peptide fragment fingerprinting (PPF) and whole protein MS for both protein identification and structural analysis of proteins.     

Rapid synthesis of titanium(IV)‐immobilized magnetic mesoporous silica nanoparticles for endogenous phosphopeptides enrichment

The MALDI‐TOF MS has already been a main platform for phosphoproteome analysis. However, there are some weaknesses in direct analysis of endogenous phosphopeptides by MALDI‐TOF MS because of the serious suppression effect and poor ionization efficiency, which is brought by the excess of nonphosphopeptides and protein. It is essential to enrich endogenous phosphopeptides from complex biosamples efficiently prior to MALDI‐TOF MS analysis. Herein, we present a time‐saving and detailed protocol for the synthesis of titanium(iv)‐immobilized magnetic mesoporous silica nanoparticles (denoted as Fe₃O₄@mSiO₂‐Ti⁴⁺), the subsequent enrichment process, and MALDI‐TOF MS analysis. We tested the LOD, size‐exclusive effect, reproducibility, and stability of Fe₃O₄@mSiO₂‐Ti⁴⁺ nanoparticles. Furthermore, the ability of this protocol for identifying endogenous phosphopeptides in healthy human serum and saliva was investigated.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

Speciation of coagulase negative staphylococci, isolated from indoor air, using SDS PAGE gel bands of expressed proteins followed by MALDI TOF MS and MALDI TOF-TOF MS-MS analysis of tryptic peptides

A group of coagulase negative staphylococcal strains isolated from indoor air of occupied school rooms were the subject of this study. Conventional MALDI TOF MS profiling of cellular extracts and physiological tests (including API STAPH) provided incomplete identification of the set of strains. After separation of a 100 kDa band using 1D gel electrophoresis, profiling of peptides (released with tryptic digestion) using MALDI TOF MS allowed improved bacterial speciation in addition to determination of the identity of the protein of origin (aconitate hydratase). This was performed by Mascot search, empirical observation and computer-generated cross-correlation analysis of environmental isolates versus reference strains. The species studied included some with sequenced genomes and others with un-sequenced genomes. Peptide sequences were confirmed to originate from aconitate hydratase using MALDI TOF-TOF MS-MS analysis of a diverse set of m/z values representing variable and conserved sequences. The methodological approach described here might have widespread application in speciation of environmental isolates of diverse origin and in identification of their expressed proteins.     

QuantiSpec — Quantitative mass spectrometry data analysis of 15N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Rapid selection of peptide containing fractions in off‐line 2‐D HPLC in shotgun proteome analysis by screening with MALDI TOF MS

A simple and fast screening method for the selection of fractions of first dimension separation to be analyzed in second dimension‐MS/MS experiments in offline multidimensional liquid chromatographic separation schemes for shotgun proteome analysis was developed. The method is based on the measurement of total peptide content of the first dimension fractions by MALDI MS and was established using a tryptic digest of a bacterial proteome. The results of the screening process were in good agreement with those obtained in a detailed proteome analysis performed by RP×ion‐pair RP‐MALDI TOF/TOF MS analysis. The method supports a straightforward planning of experiments, also enabling a reduction of overall measurement time in shotgun proteome analysis.     

Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity‐related protein fragments. After purification with surface‐active magnetic beads, MALDI‐TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC‐MALDI‐TOF/TOF and confirmed by MALDI‐FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma‐related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.     

ACE Inhibitory and Antioxidant Activities of Novel Peptides from <Emphasis Type="Italic">Scorpaena notata</Emphasis> By-product Protein Hydrolysate

This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC₅₀ values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.     

Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.     

Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein

A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in −18 Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp–Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H₂¹⁸O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without ¹⁸O incorporation. The light chain peptide L2 VTITCITSTDID₁₂DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp–Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD₂₄DFK. No other isomerizations were detected in CDR peptides containing either Asp–Ser or Asp–Thr motifs.     

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI‐TOF‐MS read‐out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI‐TOF‐MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read‐out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI‐TOF‐MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 M_r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu³¹²–Arg³²² and Arg³²³–Lys³⁵⁰) and extracellular FPR1 peptide (Ile¹⁹¹–Arg²⁰¹) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala³²³–Lys³⁵⁰ only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr³²⁵, Ser³²⁸, Thr³²⁹, Thr³³¹, Ser³³², Thr³³⁴, and Thr³³⁹. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC₅₀ = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.     

Highly selective enrichment of phosphopeptides by on‐chip indium oxide functionalized magnetic nanoparticles coupled with MALDI‐TOF MS

Selective and efficient preconcentration is indispensable for low concentration of phosphopeptides in phosphorylated protein‐related samples prior to MS‐based analysis. Herein, an on‐chip system coupled magnetic SPE with MALDI‐TOF MS was designed. A metal oxide affinity chromatography material, indium oxide, was coated on the surface of Fe₃O₄ magnetic nanoparticles to prepare the adsorbent, spatially confined with an applied magnetic field. The adsorbent exhibited high selectivity for phosphopeptides in tryptic digests of the mixture of β‐casein and BSA (1:1000) and the mixture of β‐casein, BSA, and ovalbumin (1:100:100). Thanking to the enrichment ability and specificity for phosphopeptides with the adsorbent, the on‐chip magnetic SPE‐MALDI‐TOF MS approach showed high sensitivity with a low detection limit of 4 fmol. In addition, the developed approach was used to analyze phosphopetides in non‐fat milk digests and human serum successfully.