FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091547938> ?p ?o ?g. }

• W2091547938 endingPage "30318" @default.
• W2091547938 startingPage "30315" @default.
• W2091547938 abstract "Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage. Mass spectrometry (MS)-based methods coupled to reverse phase chromatography separation are a useful technology to analyze complex peptide pools that are comprised of different peptides with unrelated sequences. In antigen presentation, proteasomes generate a set of short peptides that are closely related and overlapping and in some instances may even have identical retention times and identical masses. In these situations, micro-liquid chromatography-MS/MS focused on each theoretical parent ion followed by manual interpretation optimizes the identification of generated peptides. The results suggest that the degradation of short antigens by the proteasome occurs by sequential cleavage. Recognition of virus-infected cells by cytotoxic T lymphocytes requires prior proteolytic processing of viral proteins (1York I.A. Goldberg A.L. Mo X.Y. Rock K.L. Immunol. Rev. 1999; 172: 49-66Crossref PubMed Scopus (187) Google Scholar). This degradation generates short peptides that are translocated to the endoplasmic reticulum lumen by transporters associated with antigen processing, assembled with major histocompatibility complex class I heavy chain and β2-microglobulin, and transported to the cell membrane (1York I.A. Goldberg A.L. Mo X.Y. Rock K.L. Immunol. Rev. 1999; 172: 49-66Crossref PubMed Scopus (187) Google Scholar). In infected cells, processing predominantly takes place in the cytosol by the combined action of proteasomes and degradative peptidases. Processing generates a broad diversity of peptides including a small fraction of the correct epitope or N-terminally extended precursors (1York I.A. Goldberg A.L. Mo X.Y. Rock K.L. Immunol. Rev. 1999; 172: 49-66Crossref PubMed Scopus (187) Google Scholar) that are utilized for major histocompatibility complex class I antigen presentation. These epitope precursor peptides require N-terminal trimming by aminopeptidases either in the cytosol or in the endoplasmic reticulum (2Saveanu L. Fruci D. van Endert P. Mol. Immunol. 2002; 39: 203-215Crossref PubMed Scopus (60) Google Scholar). We are interested in the identification of the set of processed peptides generated from short viral proteins by antigen processing by the proteasome and/or other proteases. In a previous work (3López D. Del Val M. J. Immunol. 1997; 159: 5769-5772PubMed Google Scholar), we studied antigen processing from recombinant vaccinia viruses encoding two closely related minigene products encompassing murine cytomegalovirus pp89 immunodominant nonamer epitope (4Del Val M. Volkmer H. Rothbard J.B. Jonjic S. Messerle M. Schickedanz J. Reddehase M.J. Koszinowski U.H. J. Virol. 1988; 62: 3965-3972Crossref PubMed Google Scholar). By using protease inhibitors, we defined a role for proteasomes in physiological intracellular antigen processing of short antigens. One of the minigenes (m19) codes for a 19-mer peptide, MDIGAYPHFMPTNLAGDPY. It represents the immunodominant murine cytomegalovirus 9pp89 epitope (YPHFMPTNL) and the local flanking amino acids of HBe carrier protein. In addition, it presents a biterminal alanine spacer between the 9pp89 core and HBe flanking residues, which was previously reported to enhance antigenicity (5Eggers M. Boesfabian B. Ruppert T. Kloetzel P.M. Koszinowski U.H. J. Exp. Med. 1995; 182: 1865-1870Crossref PubMed Scopus (104) Google Scholar). In this classic paper Eggers et al. (5Eggers M. Boesfabian B. Ruppert T. Kloetzel P.M. Koszinowski U.H. J. Exp. Med. 1995; 182: 1865-1870Crossref PubMed Scopus (104) Google Scholar) characterized the proteasome digestion products of this m19 peptide. The authors identified two cleavage products; the 1–14 peptide (MDIGAYPHFMPTNL) as the major degradation product and also the 9pp89 epitope (6YPHFMPTNL14) were detected. As the proteasome is a multicatalytic protease with three different activities (6Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar), some authors have demonstrated a broad spectrum of processed peptides generated by the action of this protease on different proteins (reviewed in Ref. 7Kloetzel P.M. Biochim. Biophys. Acta. 2004; 1695: 225-233Crossref PubMed Scopus (152) Google Scholar). Accordingly, we can expect higher diversity in m19 cleavage experiments that are revealed with the classic MS 2The abbreviations used are: MS, mass spectrometry; LC, liquid chromatography; ESI, electrospray ionization; IT, ion trap; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. analysis (5Eggers M. Boesfabian B. Ruppert T. Kloetzel P.M. Koszinowski U.H. J. Exp. Med. 1995; 182: 1865-1870Crossref PubMed Scopus (104) Google Scholar). In the last years immunoproteomics or mass spectrometry-based methods to study the targets of the immune response have been developed (8Purcell A.W. Gorman J.J. Mol. Cell. Proteomics. 2004; 3: 193-208Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). With the standard automated micro-LC-MS/MS scan mode we detected four m19-derived cleavage products. Therefore we modified this technical approach involving micro-LC-MS/MS focused on each theoretical parental cleavage product followed by manual interpretation, and thus we optimize the detection of generated peptides. This strategy allow the identification of nine cleavage products. With these data, we suggest a sequential cleavage model for proteasome activity on the m19 peptide. Synthetic Peptides—The peptides were synthesized in an Applied Biosystems peptide synthesizer model 433A (Foster City, CA), purified, and found homogeneous by HPLC analysis. HPLC Analysis of Peptides—Five nanomoles of each synthetic peptide was analyzed by reverse phase HPLC (SMART system equipped with a μRPC Sephasil C18 SC 2.1/10 column (GE Healthcare)). The eluents used were: A, 19.6% acetonitrile containing 0.1% trifluoroacetic acid; B, 70% acetonitrile containing 0.1% trifluoroacetic acid. The gradient was 0–31.2% B in 18 min and 31.2–100% in 4 min, and the flow rate was 210 μl/min–1. Several runs were performed with each synthetic peptide, and their location inside the acetonitrile gradient was reproducible with a maximum error of one fraction (52 μl). Proteasome Assays—Isolation and purification of 20 S proteasomes from rabbit psoas muscle used were performed exactly as previously described (9Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). The reaction conditions for proteasome were as published (10Kisselev A.F. Garcia-Calvo M. Overkleeft H.S. Peterson E. Pennington M.W. Ploegh H.L. Thornberry N.A. Goldberg A.L. J. Biol. Chem. 2003; 278: 35869-35877Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Mass Spectrometry Analysis—MALDI-TOF mass spectrometry was performed in a Reflex IV instrument (Brucker-Franzen Analytik, Bremen, Germany) operating in the positive ion reflection mode. Twenty-five microliters of sample were dried, resuspended in 1 μl of 0.1% trifluoroacetic acid in a 2:1 solution of water:acetonitrile, and mixed with 1 μl of saturated α-cyanohydroxycinnamic acid matrix in the same solution. One microliter of the mixture was dried and subjected to analysis. Aliquots of total digestions were dried and dissolved in 10 μl of 0.5% acetic acid in water and sequenced by quadrupole ion trap micro-HPLC (Biobasic C18 column, 150 × 0.18 mm (Thermo Electron, San Jose, CA) electrospray MS/MS in a Deca XP LCQ mass spectrometer (Thermo Electron). The eluents used were: A, 0.5% acetic acid in water; B, 80% acetonitrile containing 0.5% acetic acid. The gradient was 0–40% B in 24 min and 40–100% in 5 min, and the flow rate was 1.5 μl/min–1. We used the MS/MS mode focused to each hypothetical parental peptide with a isolation width (m/z) of 1.5 Da. The charge and the mass of the ionic species were determined by high resolution sampling of the mass/charge rank. Collision energy and ion precursor resolution were improved to optimize the fragmentation spectrum. As a first step, proteasomes were isolated and purified (9Kisselev A.F. Akopian T.N. Woo K.M. Goldberg A.L. J. Biol. Chem. 1999; 274: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Next, m19 synthetic peptide was incubated with purified proteasomes. After that, the cleavage products generated by this protease involved in m19 processing were analyzed by mass spectrometry. In a first approximation MALDI-MS analysis of total digestion was carried out. This system allowed the detection of three different peptidic species derived from the m19 sequence (Fig. 1). Species number 3 had identical m/z with the 1–14 peptide detected as the major cleavage product by Eggers et al. (5Eggers M. Boesfabian B. Ruppert T. Kloetzel P.M. Koszinowski U.H. J. Exp. Med. 1995; 182: 1865-1870Crossref PubMed Scopus (104) Google Scholar). To elucidate the nature of the two additional products detected and verify the nature of species number 3, MS/MS analysis was performed. To this end, micro-HPLC with on-line detection by ESI-IT MS/MS was performed. This strategy in addition to the identification of peptide by fragmentation also increases the resolution of peptide separation before detection and subsequently enhances the sensitivity of the assay. In this automated separation-detection system, four peptidic products were unequivocally identified: the three previously detected by MALDI mass spectrometry together with one new shorter peptide (Fig. 2, top). In addition, uncertainties on the nature of species numbers 1 and 2, inherent to previous MALDI analysis, were resolved. In summary, 1–14, 4–14, 5–14, and 6–14 peptides were identified as cleavage products. No peptides were detected that arose from cleavages within the 9pp89 core in both MALDI-TOF and ESI-IT analysis.FIGURE 2Digestion pattern of m19 synthetic peptide with purified proteasome analyzed by ESI-IT mass spectrometry. The sequence of the cytomegalovirus epitope is boxed. The lines show peptide products identified by MS analysis, and their respective name is indicated. The peptide products identified by micro-HPLC with on-line detection by ESI-IT MS/MS analysis were depicted above the m19 amino acid sequence. Similarly, the micro-HPLC-MS/MS data using analysis of the predefined parent ion were depicted below the m19 amino acid sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Usually the automated MS/MS scan mode allows for the identification of predominant parental peptidic species, but we are interested in the identification of the largest spectrum of products generated by the proteasome activity. The potential digestion products of m19 are closely related and the collection is highly complex. Indeed, in the mixture of digestion products of m19 peptide by the proteasome 35 theoretical different peptides are possible that contain the cytomegalovirus 6–14 nonamer core, that is that are immunologically relevant. As the m19 sequence presents two Met (easily oxidized) located at the N terminus and at residue number 10, all processed relevant peptides could appear also as the oxidized molecular ion. In addition, five of these peptides present the two Met residues in their sequence. In summary, we need to detect 42 distinct molecular ions that correspond to 80 relevant molecular species derived from the m19 sequence; some of them have the same hydrophobic properties and some have identical daughter ions produced by MS fragmentation. As the hypothetical cleavage products are overlapping peptides because they have an identical nonamer core and present staged length, their hydrophobic properties in reverse phase HPLC could be related. In addition, the two residues flanking the 9pp89 epitope are symmetrical, and thus, their corresponding peptides (A9pp89 and 9pp89A or GA9pp89 and 9pp89AG) present identical HPLC retention properties as well as m/z values. Previous HPLC separation analysis (data not shown) indicated a short separation of a maximum of 4 to 5 fractions between the two extreme peptides, m19 and 9pp89 epitope (6YPHFMPTNL14). This is true even when the gradient used for elution of the synthetic peptides from the reverse phase HPLC column was rather flat (see “Experimental Procedures”). Thus, several overlapping peptides could co-elute in the same HPLC fraction. A general limitation of the analysis of complex mixtures by MS is the inability to discriminate isomers because MS/MS data are often ambiguous when multiple components are fragmented simultaneously. This might explain the limited improvement obtained with HPLC-ESI-IT over MALDI-MS analysis. Thus, we studied each one of the 35 theoretical different peptides that contain the cytomegalovirus 6–14 nonamer core in successive micro-HPLC assays to explore all possible products of the proteasome activity. Because only ions of selected mass-to-charge ratio are monitored, their scan mode generally provides higher sensitivity than does the full scan mode. Thus, by using micro-LC-MS/MS with fixed precursor to analyze each theoretical doubly charged ion derived from m19 sequence and performing a continuous operation in MS/MS under standard fragmentation conditions, we can acquire more information on the studied biological process. Fig. 3 shows the interpretation of overlapping products derived from the m19 substrate. Five different molecular ions could be detected by MS/MS fragmentation of the doubly charged ion at m/z 810.9. This is the most complex indecisiveness that exists among all potential overlapping peptides derived from the m19 sequence. This m/z corresponds to five different 14-mers: the 6–19 peptide, both 2–16 and 3–17 peptides with their respective oxidized Met, and the 1–14 peptide with either Met-1 or Met-10 oxidized. As shown in the inset of Fig. 3, the mass spectrometer detects a molecular ion with this m/z range around minute 36 of the HPLC gradient compatible with the five molecular species cited above. The fragment spectrum obtained was manually interpreted, yielding two different sequences that satisfactorily matched the pattern of fragment ions. This analysis discards the presence of three of five candidates and as shown in Fig. 3 demonstrates the existence of both 1–14 peptides with either Met-1 or Met-10 oxidized. Fig. 2 (bottom) summarizes the results obtained with the micro-HPLC-MS/MS operating with the parental ion fixed for all isomeric peptide mixtures. The MS/MS fragmentation spectra were manually interpreted to identify all co-eluting overlapping peptides including those present in lower relative amounts. Some peptides such as 1–17 were found in all cleavage experiments including the negative control with only the m19 synthetic peptide without proteases and were not considered. These results agree with some reports (11Skribanek Z. Mezo G. Mak M. Hudecz F. J. Pept. Sci. 2002; 8: 398-406Crossref PubMed Scopus (17) Google Scholar) that indicate that Asp-Pro bonds of synthetic peptides can undergo spontaneous cleavage under acid conditions such as those used in solubilization and purification of peptides prior to ESI-IT analysis. These data show a broad spectrum of processed peptides including the four previously defined (see above, Fig. 2) and demonstrate that the proteasome generates a collection of peptides where most peptides were N-extended and had the correct C-terminal Leu. Altogether we suggest the sequential cleavage model depicted in Fig. 4 as the major pathway followed by the proteasome. First, the proteasome would cut the m19 peptide between residues Gly-16 and Asp-17. In a second step, the two C-terminal residues (Ala-15 and Gly-16) of this product would be removed. Next, N-terminal Met and Asp residues would be cleaved. Afterward, the N terminus of this 3–19 product would continue to be trimmed residue by residue. This model accounts for three-fourths of the total amount of detected peptides. It has been proposed that the substrate interacts with the large inner surface of the proteasome core at undefined non-catalytic sites (12Kisselev A.F. Kaganovich D. Goldberg A.L. J. Biol. Chem. 2002; 277: 22260-22270Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In addition, some authors suggest that the substrate can be covalently attached to one active site in a transition state complex and then be attacked in turn by other active sites (13Lowe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1381) Google Scholar). Therefore, as the 1–14 peptide is the major cleavage product of m19 degradation (Fig. 1) and we detect mainly N-terminal step by step trimming, this model suggests that Leu-14 is fixed whereas adjacent catalytic sites remove sequentially these N-terminal residues. These N-extended peptides could be trimmed by aminopeptidases (2Saveanu L. Fruci D. van Endert P. Mol. Immunol. 2002; 39: 203-215Crossref PubMed Scopus (60) Google Scholar). In addition to cleavages inside the 9pp89 epitope, the different prediction algorithms for proteasome activity 3D. López, O. Calero, M. Jiménez, M. García-Calvo, and M. Del Val, unpublished data. predict the C-terminal cleavage but not the N-end trimming observed in our experiments. Alternatively, in each m19 molecule the proteasome may simultaneously perform two cleavages, one with relaxed specificity at the N terminus and the other one strongly focused on the preferred cleavage site after Leu-14. With three other additional products detected (4–15, 2–16, and 3–17 peptides) no obvious specificity patterns were found. The most simple explanation could be that some m19 molecules were cut with a relaxed specificity in the two cleavages that generates these individual peptides independently. In summary, our results show how the identification of highly related peptidic species in a highly selective and specific manner is possible and allow its application to elucidate the mechanisms of antigen processing. We thank Dr. A. F. Kisselev for proteasome isolation and purification." @default.
• W2091547938 created "2016-06-24" @default.
• W2091547938 creator A5006517831 @default.
• W2091547938 creator A5027241622 @default.
• W2091547938 creator A5028354833 @default.
• W2091547938 creator A5030848884 @default.
• W2091547938 creator A5063263984 @default.
• W2091547938 date "2006-10-01" @default.
• W2091547938 modified "2023-09-26" @default.
• W2091547938 title "Antigen Processing of a Short Viral Antigen by Proteasomes" @default.
• W2091547938 cites W1526952883 @default.
• W2091547938 cites W1975460230 @default.
• W2091547938 cites W1978131501 @default.
• W2091547938 cites W1982186676 @default.
• W2091547938 cites W2027125822 @default.
• W2091547938 cites W2085760247 @default.
• W2091547938 cites W2093686252 @default.
• W2091547938 cites W2103465870 @default.
• W2091547938 cites W2106906885 @default.
• W2091547938 cites W2131043426 @default.
• W2091547938 cites W2142455478 @default.
• W2091547938 cites W2170402078 @default.
• W2091547938 cites W2178503716 @default.
• W2091547938 doi "https://doi.org/10.1074/jbc.m605973200" @default.
• W2091547938 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16861221" @default.
• W2091547938 hasPublicationYear "2006" @default.
• W2091547938 type Work @default.
• W2091547938 sameAs 2091547938 @default.
• W2091547938 citedByCount "11" @default.
• W2091547938 countsByYear W20915479382015 @default.
• W2091547938 countsByYear W20915479382016 @default.
• W2091547938 crossrefType "journal-article" @default.
• W2091547938 hasAuthorship W2091547938A5006517831 @default.
• W2091547938 hasAuthorship W2091547938A5027241622 @default.
• W2091547938 hasAuthorship W2091547938A5028354833 @default.
• W2091547938 hasAuthorship W2091547938A5030848884 @default.
• W2091547938 hasAuthorship W2091547938A5063263984 @default.
• W2091547938 hasBestOaLocation W20915479381 @default.
• W2091547938 hasConcept C108249834 @default.
• W2091547938 hasConcept C147483822 @default.
• W2091547938 hasConcept C159047783 @default.
• W2091547938 hasConcept C170627219 @default.
• W2091547938 hasConcept C185592680 @default.
• W2091547938 hasConcept C203014093 @default.
• W2091547938 hasConcept C207936829 @default.
• W2091547938 hasConcept C86803240 @default.
• W2091547938 hasConceptScore W2091547938C108249834 @default.
• W2091547938 hasConceptScore W2091547938C147483822 @default.
• W2091547938 hasConceptScore W2091547938C159047783 @default.
• W2091547938 hasConceptScore W2091547938C170627219 @default.
• W2091547938 hasConceptScore W2091547938C185592680 @default.
• W2091547938 hasConceptScore W2091547938C203014093 @default.
• W2091547938 hasConceptScore W2091547938C207936829 @default.
• W2091547938 hasConceptScore W2091547938C86803240 @default.
• W2091547938 hasIssue "41" @default.
• W2091547938 hasLocation W20915479381 @default.
• W2091547938 hasLocation W20915479382 @default.
• W2091547938 hasOpenAccess W2091547938 @default.
• W2091547938 hasPrimaryLocation W20915479381 @default.
• W2091547938 hasRelatedWork W1527215315 @default.
• W2091547938 hasRelatedWork W1968866496 @default.
• W2091547938 hasRelatedWork W1979465056 @default.
• W2091547938 hasRelatedWork W2069615176 @default.
• W2091547938 hasRelatedWork W2076922394 @default.
• W2091547938 hasRelatedWork W2220102944 @default.
• W2091547938 hasRelatedWork W2245876498 @default.
• W2091547938 hasRelatedWork W2414745257 @default.
• W2091547938 hasRelatedWork W62304571 @default.
• W2091547938 hasRelatedWork W2080067311 @default.
• W2091547938 hasVolume "281" @default.
• W2091547938 isParatext "false" @default.
• W2091547938 isRetracted "false" @default.
• W2091547938 magId "2091547938" @default.
• W2091547938 workType "article" @default.