An antibody to a synthetic peptide recognizes polyomavirus middle-T antigen and reveals multiple in vitro tyrosine phosphorylation sites.

Antibodies were raised against three synthetic peptides corresponding to sequences surrounding tyrosine 315, a putative in vitro phosphorylation site in polyomavirus middle-T antigen. Only one of the peptides (called C and corresponding to residues 311 to 330) elicited antibodies that recognized middle-T efficiently. Middle-T present in immunoprecipitates formed with purified anti-C serum still accepted phosphate on tyrosine in an in vitro kinase reaction. This implies that tyrosines other than 315 and 322 that lie within the antibody binding region are phosphorylated under these conditions. This conclusion was supported by the altered partial V8 proteolysis fingerprint of the labeled middle-T. Two-dimensional tryptic fingerprint analysis of 32P-labeled middle-T showed that several tryptic peptides identified as including tyrosine 315 and 322 were missing from middle-T labeled in anti-C immunoprecipitates compared with middle-T labeled in immunoprecipitates made by using anti-tumor cell serum. However, one major labeled peptide remained. This peptide was also present in fingerprints of 32P-labeled middle-T coded by M45, dl23, pAS131, and dl1013, but a peptide with altered mobility was present in dl8 middle-T. This identified the peptide as including tyrosine 250. We deduce from these data that (i) the presence of the antibody against peptide C inhibits phosphorylation of tyrosines 315 and 322; (ii) middle-T labeled in the kinase reaction after immunoprecipitation with anti-C serum is phosphorylated on tyrosine 250; and (iii) when anti-tumor cell serum is used in the in vitro kinase reaction, middle-T is phosphorylated at multiple sites, including residues 250, 315, and 322.     

Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS

Antibodies directed against the C-terminus of cholecystokinin octapeptide (CCK8) and caerulein were used to study immunoreactive peptides in pig brain. One antibody, a mouse monoclonal raised to caerulein (c.MAb), reacts equally with heptadecapeptide gastrin (G17), CCK8 and caerulein, the other raised to CCK8 (L48) shows 10 times lower immunoreactivity with caerulein compared with G17 and CCK8. Extracts were purified by adsorption to alginic acid, gel filtration chromatography and reversed phase HPLC. In addition to material with the expected properties of CCK33, 39 and 58 a novel peptide was identified that reacted 10 times better with c.MAb compared with L48. This material emerged in a similar position to CCK58 on Sephadex G50 but had a greater retention time on reversed phase HPLC. It had CCK-like bioactivity and digestion with trypsin gave a fragment showing a pattern of immunoreactivity similar to that of the parent compound. This pattern of activity is distinct from other known mammalian CCKs; the material may represent an addition to the gastrin-CCK family in mammals.     

Multiple gastrin-releasing peptide gene-associated peptides are produced by a human small cell lung cancer line

Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.     

Continuous flow peptide synthesis on aminopolyoxyethylene-polystyrene graft copolymer using Fmoc-strategy

An insoluble graft copolymer consisting of the covalently bound polyoxyethylene to cross-linked polystyrene (HO-POE-PS) was prepared by anionic polymerization of ethylene oxide on the resin. The copolymer was then converted to the corresponding amino-polymer (H2N-POE-PS) and the latter was employed as the solid carrier for peptide synthesis. Although HO-POE-PS has successfully been employed as a carrier for peptide synthesis by the standard shaking procedure using t-butoxycarbonyl-amino acids, now we deemed it of interest to test its suitability for the continuous flow synthesis. Thus, the C-terminal octapeptide of the porcine insulin B chain (B23-30) was prepared by this procedure using a photolabile anchoring group and fluoren-9-ylmethoxycarbonyl-amino acids. All the reactions were carried out in a continuous flow manner in a steel column under pressure using a high-performance liquid chromatography (HPLC) system. At the end of the synthesis, a sample of the protected peptide was cleaved from the support by photolysis. For the cleavage of another sample, an aqueous solution of sodium carbonate was employed. The protected peptide was purified on silica gel and Sephadex-LH 20. All the protecting groups of a sample of the octapeptide were removed with piperidine/dimethylformamide and trifluoroacetic acid and the deblocked peptide was purified by ion-exchange chromatography. The free peptide was shown to be homogeneous by thin-layer chromatography, HPLC, and electrophoresis. The identify of the free octapeptide was confirmed by amino-acid analysis, 13C-nuclear magnetic resonance measurement and field-desorption mass spectrometry. The peptide was also shown to be free of racemization.     

Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein

N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids.     

Vaccination with tumor peptide in CpG adjuvant protects via IFN-gamma-dependent CD4 cell immunity

The low frequency of tumor Ag-specific T cells in vivo has made it challenging to directly measure their clonal sizes and cytokine signatures. We used a new generation ELISPOT approach to study the constitutive immunogenicity of the RMA tumor in syngeneic B6 mice and adjuvant-guided immunity against an MHC class II-restricted RMA peptide, H11.1. The RMA tumor was found to activate cells of the innate immune system and to induce a type 1 polarized, RMA-specific CD4 and CD8 T cell response. With clonal sizes approximately 10/10(6), the magnitude of this constitutively induced immune response did not suffice to control the tumor cell growth. In contrast, immunization with H11.1 peptide, using an immunostimulatory CpG oligonucleotide or CFA as adjuvant, engaged approximately 25- or approximately 10-fold higher clonal sizes of type 1 polarized CD4 cells, respectively. Therefore, the CpG oligonucleotide functioned as a stronger type 1 adjuvant and, unlike CFA, elicited protective immunity. The protection was IFN-gamma dependent, as it was not inducible in IFN-gamma knockout mice. Therefore, CpG adjuvant-guided induction of type 1 immunity against tumor Ags might be a promising subunit vaccination approach.     

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.     

Identification of a novel tumor-specific CTL epitope presented by RMA, EL-4, and MBL-2 lymphomas reveals their common origin

C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR alpha- and beta-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.     

HSP70 natively and specifically associates with an N-terminal dermcidin-derived peptide that contains an HLA-A*03 antigenic epitope

Tumor cells very often have elevated expression of HSP70, the anti-apoptotic properties of which contribute to overall tumor survival. Independent of its anti-apoptotic properties, HSP70 was also suggested to be involved in the antigen presentation process by chaperoning cytosolic peptides, thus protecting them from rapid degradation and securing the peptide pool for further processing. In this study, we identified a 33-amino acid N-terminal dermcidin (DCD)-derived peptide from the repertoire of in vivo HSP70-associated peptides isolated from a leukemic cell line, K562. The DCD peptide has been previously shown to be involved in tumorigenesis, to increase tumor survival rate, to improve tumor stress resistance, and to aid growth. We show that HSP70 is a specific binding partner for the DCD prosurvival peptide and define an ATP-dependent DCD-binding site (GNPCH). We also identify an HLA-A*03 antigenic epitope within the DCD peptide, which follows and partially overlaps the HSP70-binding site (CHEASAAQK). This study describes the interaction between HSP70 and the DCD-derived prosurvival peptide, an interaction that may direct the peptide toward antigen presentation and independently contribute to the prosurvival mechanism mediated by DCD.     

"Joining peptide" of pro-opiomelanocortin. II. Interspecies heterogeneity of the joining peptide fragment

The use of an antiserum raised against the joining peptide sequence -23 to -14 of bovine pro-opiomelanocortin (POMC) enabled the detection of related immunoreactive sequences of peptides in bovine, porcine, mouse and guinea-pig pituitaries, as well as in mouse brain and cerebral cortex, guinea-pig cerebral cortex, and bovine hypothalamus. Gel chromatography of pituitary extracts (Sephadex G-75 and Bio-Gel P-4) indicated the presence of several immunoreactive joining peptide fragments ranging in the molecular weight range (Mr) of 1,500 to 2,300. Furthermore, high molecular weight (Mr greater than 22,500) immunoreactive-precursor from bovine anterior pituitary was readily digested with trypsin into an immunoreactive fragment of approximately Mr 1,500. Analyses of these immunoreactive peptides by reverse-phase high-performance liquid chromatography (HPLC) led to their resolution into six distinct peptides. The only apparent correspondence in the elution profiles of immunoreactive peptide profiles between different mammalian species was the identification of a similar fragment (Mr 2,000) from bovine and guinea-pig pituitaries. Thus, we conclude that immunoreactivity to the joining peptide region of POMC from various mammalian species exhibits a degree of heterogeneity in its composition. The relatively low levels of immunoreactivity in comparison to that of ACTH also suggest that the joining peptide domain may be further processed. The hormonal status of the joining peptide region remains to be determined.     

Metorphamide, a C-terminally amidated opioid peptide, in human adrenal and human phaeochromocytoma

There appears to be only one possible site for the production of an amidated peptide in the human proenkephalin sequence; this will give rise to the peptide named metorphamide. Since amidation of peptides is commonly an activation step in the synthesis of regulatory peptides, we have examined the levels and form of immunoreactivity to metorphamide in human post-mortem adrenal and phaeochromocytoma extracts. In three out of four post-mortem adrenal extracts, and in each of the two phaeochromocytoma extracts examined, there was 3-4 times more immunoreactivity to the carboxy-terminus of pro-enkephalin, Met-enkephalin(Arg6,Phe7), than to metorphamide. The metorphamide immunoreactivity was shown in each extract to measure only the amidated octapeptide according to gel exclusion and reverse-phase chromatography data. The implications for processing of proenkephalin in human adrenal are indicated.     

Functional characterization of CTL against gp100 altered peptide ligands

In this study, four modified gp100 peptides were designed by combining amino acids from the melanoma peptide antigen gp100((209-217)) with preferred primary and auxiliary HLA-A *0201 anchor residues previously identified from combinatorial peptide library screening with recombinant HLA-A*0201. These modified peptides demonstrated stronger binding affinity for the HLA-A*0201 molecule compared to wild-type gp100 peptide. Nine CTL lines generated from patients immunized with the g209-2 M peptide and one CTL line from a non-immunized patient were tested for the ability to respond to these modified gp100 peptides. Stimulation of CTL by two of four modified peptides induced higher levels of IFN-gamma secretion than the wild-type gp100 peptide, demonstrating that higher peptide binding affinity for HLA molecules does not necessarily equate to functional activity of CTL. Two major and one minor CTL recognition pattern were observed, irrespective of previous peptide immunization, suggesting that multiple, rationally designed modified tumor peptides for the same epitope stimulate a broad CTL response by activating multiple CTL capable of cross-reacting with the natural antigenic peptide.     

Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region

The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.     

Intratumoral peptide injection enhances tumor cell antigenicity recognized by cytotoxic T lymphocytes: a potential option for improvement in antigen-specific cancer immunotherapy

Antigen-specific cancer immunotherapy is a promising strategy for improving cancer treatment. Recently, many tumor-associated antigens and their epitopes recognized by cytotoxic T lymphocytes (CTLs) have been identified. However, the density of endogenously presented antigen-derived peptides on tumor cells is generally sparse, resulting in the inability of antigen-specific CTLs to work effectively. We hypothesize that increasing the density of an antigen-derived peptide would enhance antigen-specific cancer immunotherapy. Here, we demonstrated that intratumoral peptide injection leads to additional peptide loading onto major histocompatibility complex class I molecules of tumor cells, enhancing tumor cell recognition by antigen-specific CTLs. In in vitro studies, human leukocyte antigen (HLA)-A*02:01-restricted glypican-3_144–152 (FVGEFFTDV) and cytomegalovirus_495–503 (NLVPMVATV) peptide-specific CTLs showed strong activity against all peptide-pulsed cell lines, regardless of whether the tumor cells expressed the antigen. In in vivo studies using immunodeficient mice, glypican-3_144–152 and cytomegalovirus_495–503 peptides injected into a solid mass were loaded onto HLA class I molecules of tumor cells. In a peptide vaccine model and an adoptive cell transfer model using C57BL/6 mice, intratumoral injection of ovalbumin_257–264 peptide (SIINFEKL) was effective for tumor growth inhibition and survival against ovalbumin-negative tumors without adverse reactions. Moreover, we demonstrated an antigen-spreading effect that occurred after intratumoral peptide injection. Intratumoral peptide injection enhances tumor cell antigenicity and may be a useful option for improvement in antigen-specific cancer immunotherapy against solid tumors.     

New peptide inhibitors of type IB topoisomerases: similarities and differences vis-a-vis inhibitors of tyrosine recombinases

Topoisomerases relieve topological tension in DNA by breaking and rejoining DNA phosphodiester bonds. Type IB topoisomerases such as vaccinia topoisomerase (vTopo) and human topoisomerase I are structurally and mechanistically similar to the tyrosine recombinase family of enzymes, which includes bacteriophage lambda Integrase (Int). Previously, our laboratory identified peptide inhibitors of Int from a synthetic peptide combinatorial library. The most potent of these peptides also inhibit vTopo. Here, we used the same mixture-based screening procedure to identify peptide inhibitors directly against vTopo using a plasmid relaxation assay. The two most potent new peptides identified, WYCRCK and KCCRCK, inhibit plasmid relaxation, DNA cleavage and Holliday junction (HJ) resolution mediated by vTopo. The peptides tested bind double-stranded DNA at high concentrations but do not appear to displace the enzyme from its DNA substrate. WYCRCK binds specifically to HJ and perturbs the central base-pairing. This peptide also accumulates HJ intermediates when it inhibits Int-mediated recombination, whereas KCCRCK does not. Interestingly, WYCRCK shares four amino acids with a peptide identified against Int, WRWYCR. The octapeptide WRWYCRCK, containing amino acids from both hexapeptides, is more potent than either against vTopo. All peptides are less potent against the type IA Escherichia coli topoisomerase I or against restriction endonucleases. Like the Int-inhibitory peptide WRWYCR, WYCRCK binds to HJs, and both inhibit junction resolution by vTopo. Our results suggest that the newly identified WYCRCK and peptide WRWYCR interact with a distorted DNA intermediate arising during vTopo-mediated catalysis, or interfere with specific interactions between vTopo and DNA.     

Isolation, structures, and biologic activity of neurotensin-related peptides generated in extracts of avian tissue

Two immunoreactive neurotensin-related peptides generated by the action of endogenous protease(s) on protein substrates during acid extraction of avian tissues have been isolated from extracts of turkey skin and proventriculus. One was identified as the pentadecapeptide, H-Phe-Glu-Arg-Phe-Gln-Gly-Met-Arg-Thy-Arg-Gly-Pro-Tyr-Phe-Leu-OH and the other was its C-terminal octapeptide fragment. Each peptide showed partial homology to the C-terminal, biologically active region of avian neurotensin, and isolated preparations displayed pharmacologic activity at submicromolar concentrations. Synthetic preparations were shown to be indistinguishable from the native peptides during high pressure liquid chromatography (HPLC) and bioassay. Analysis by HPLC indicated that similar peptides could be generated in extracts of proventriculus, pancreas, small intestine, skin, heart, lung, and skeletal muscle. These results, establishing the presence of a neurotensin-related sequence which can be liberated from protein(s) by the action of tissue enzyme(s), suggest that peptide(s) similar to neurotensin may be rapidly formed in order to promote physiologic regulation in multiple tissue(s).     

Immuno-affinity purification of specific antibodies against human gastrin releasing peptide (h-GRP) by the h-GRP(1-8)-linked polydimethylacrylamide resin

A new method for immuno-affinity purification of specific antibodies against human gastrin releasing peptide(h-GRP) was developed. The antiserum GP(No. 6201) elicited by h-GRP-BSA conjugate was heterogeneous and reacted not only with h-GRP and its fragments but also partially with other structurally related peptides, such as other GRPs (porcine, canine, and chicken), bombesin, and neuromedin-C. To obtain specific antibodies against human GRP, antiserum GP was purified by column chromatography on the amino-terminal octapeptide h-GRP(1-8)-linked polydimethylacrylamide resin. The antibody thus obtained was highly specific to amino-terminal sequence of h-GRP and hardly reacted with other GRPs (porcine, canine and chicken), bombesin, and even carboxy-terminal h-GRP fragments in ELISA.     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the nucleotide binding site by the substrate photoaffinity analogue [gamma-32P]-8-azido-dATP. The alpha and beta polypeptides of photolabeled terminal transferase were resolved by high-performance liquid chromatography. The beta polypeptide was digested with trypsin and fractionated by reverse-phase chromatography. Two 32P-containing fractions were isolated and subjected to amino acid sequence analysis. Peptides were identified as Ile209-Lys232 (B26) and Val233-Lys239 (B27). Peptide B26 was further resolved into two overlapping species; one contained an additional lysine residue at the N-terminus which resulted from tryptic cleavage between Lys207 and Lys208. In order to ensure that the sequenced peptides corresponded to the photolabeled species, we devised an anion-exchange procedure to isolate photolabeled peptides from the mixture. Analysis of photolabeled peptides from terminal transferase alpha beta using DEAE-cellulose chromatography followed by reverse-phase HPLC confirmed that the photolabeled species were peptides B26 and B27. Peptide B26, the major photolabeled species, contained a conserved octapeptide region found in several eucaryotic DNA polymerases. In addition, peptide B27 was flanked by a sequence that has been implicated in triphosphate binding in other proteins. Structure predictions, based on sequence data, place the two peptides identified by photolabeling in spatial proximity consistent with the participation of both in the nucleotide binding domain.     

Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discovery

Peptides have a role in the inflammatory response, tumor biology, and endocrine processes, presenting them as appealing biomarker candidates. However, peptide extraction efficacy for clinical profiling remains a pivotal technological challenge, as maximum coverage of the plasma peptidome is limited by a range of factors including the inherent complexity of human plasma and the lower concentration of peptides compared to abundant proteins. The aim of this study was to evaluate commonly employed peptide extraction methodologies in terms of total number of peptides detected and the mass range of peptides observed by MALDI. Despite showing coelution of proteins, solid-phase extraction (SPE) methods exhibited superior plasma peptide recovery than ultrafiltration, acetonitrile (ACN) precipitation, or size-exclusion chromatography methods under conditions employed in the study. Not surprisingly, in line with studies challenging the veracity of many peptide biomarker studies, the majority of identified peptides eluted from SPE methods corresponded to proteolytic truncations of the most abundant plasma proteins. The prefractionation of plasma with acetonitrile precipitation prior to SPE provided distinct ion signal profiles and is worthy of further study. In conclusion, this study favors the use of SPE in peptide extraction protocols for increased biomarker coverage and diversity from the plasma peptidome.