The 1997 ABRF Mass Spectrometry Committee Collaborative Study: Identification of Phosphopeptides in a Tryptic Digest of Apomyoglobin
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1 The 1997 ABRF Mass Spectrometry Committee Collaborative Study: Identification of Phosphopeptides in a Tryptic Digest of Apomyoglobin Kristine Swiderek, Farzin Gharahdaghi, Lowell Ericsson, Murray Hackett, Beth Fowler, Gary Hathaway, Rachel O. Loo, Rich Johnson and John Stults Beckman Research Institute at City of Hope, Duarte, CA 91010, Rockefeller University, New York, NY 10021, University of Washington, Seattle, WA 98195, AutoImmune, Inc., Lexington, MA 02173, California Institute of Technology, Pasadena, CA 91125, University of Michigan, Ann Arbor, MI 48109, Immunex, Seattle, WA 98040, Genentech, South San Francisco, CA 94080
2 INTRODUCTION The Association of Biomolecular Resource Facilities (ABRF) sponsors the activities of a number of research committees, including a Mass Spectrometry Committee. Among other interests, this committee has carried out collaborative interlaboratory studies of blind-coded test samples in order to provide participating laboratories with feedback regarding their use of mass spectrometry. The 1997 Collaborative Study was designed to test the ability of member labs to use mass information for identification of potential regulatory sites in proteins. The test sample consisted of 500 picomoles of horse apomyoglobin tryptic digest also containing equimolar amounts of two synthetic phosphorylated peptides, based on analogous portions of the apomyoglobin sequence, LFTGHPET(p)LEK and AS(p)EDLK. Participants were asked to identify the protein, identify the phosphorylated peptides, and if they desired, to further analyze the phosphopeptides using whatever approach was deemed appropriate, including molecular weight determination, and automated or manual interpretation of tandem mass spectrometry data. Participants employed a wide variety of instrumentation and methods, including MALDI TOF/PSD, ESI-TOF, triple quadrupole and ion trap mass spectrometry, coupled in most cases with a database search. The results returned by 28 participating laboratories are summarized.
3 EXPERIMENTAL AS(p)EDLK and LFTGHPET(p)LEK were synthesized and quantitated (AAA) at the Protein/DNA Technology Center (The Rockefeller University), while the tryptic digest of horse heart myoglobin was provided by the Department of Biochemistry, University of Washington. Techniques exploited for selective phosphopeptide detection on triple quadrupole instruments included precursor ion scans, stepped collision energy scans combined with on-line separations, and neutral loss scans. Precursor and stepped collision energy scans are generally applicable to Ser, Thr, and Tyr phosphorylation, while neutral loss scans favor phosphorylation at Ser and Thr. An Fe3+ affinity membrane (Pierce Spinzyme #29521) was employed to selectively extract phosphopeptides. Laboratories primarily employed Sequest, or and for identification of the unknown protein. MALDI analyses employed HCCA (alpha-cyano-4-hydroxy cinnamic acid) or DHB (2,5- dihydroxybenzoic acid). Sequence information was derived from CID spectra or carboxypeptidase digestion. Alkaline phosphatase treatment was combined with MALDI to confirm phosphorylation.
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8 ESI RESULTS Laboratory ID Laboratory ID Separation Analyzer ID Phosphopeptide Identified 0 2 l yes yes l (1,2,3,5) (8) yes yes yes (35) 03 l yes yes l (2) (8) yes yes yes? (35) 04 l l yes yes l (2) (8) yes yes yes (22) 05 l yes yes l (2) (11) yes yes yes (22) 06 l l yes yes l (2) (11,12) yes yes yes? (22) 07 l yes yes l (2) (8,20) yes yes yes (22) (22, 23,24) 08 l l yes yes l (5) (11) yes yes yes (22,23,24) 09 l yes yes l (2) (16) yes yes yes (25) l yes yes l (7) (11) yes yes yes? (22) 11 l yes yes l (8) yes yes yes (22) 12 l no no l (2) (8,13,19) yes yes (26) no (23,27) 13 l yes yes l (6) (10) yes yes no (23,27) (22) 14 l yes yes l (1,2,3) (13,14) yes yes (26?) yes (26?)? (22) 16 l yes yes l (1) (9) yes yes (26,28) no l yes yes l (2,7) (11) yes yes yes l yes yes l (2) (9) yes yes no l yes yes l (1,4) (9) yes no no some some l yes yes l (3,21) (15) yes yes no (29) l l yes yes l (2) (10,17,18) yes yes no (22) 22 l yes yes l (2) (8) yes yes yes (22) 28 l yes no l (1) (9) yes yes (26) yes (31)
9 MALDI RESULTS Laboratory ID Laboratory ID Separation ID Phosphopeptide Identified 01 l l yes some HCCA, DHB (2) (11) yes yes (32) no l yes yes HCCA, DHB (2) (8) yes yes yes (ESI) 84 ESI l by ESI HCCA (2) (8) yes yes (26) ESI ESI ESI ESI l yes yes HCCA (2) (11) yes yes ESI 90 ESI l by ESI HCCA (7) (11) yes yes (26) ESI 54 ESI l by ESI HCCA (8) yes ESI ESI 80 ESI ESI l HCCA, SA yes ESI no 80 ESI ESI l yes no HCCA (7) (11) yes no no (29) 20 l HCCA (?) (3,21) (15) yes yes (26) no 92 ESI (29) 23 l l l HCCA (7) (11) yes yes (26) no l l big only HCCA (7) (11) yes yes no (33) l yes no HCCA (2) (8) no no no? 6-8 < l yes yes HCCA (7) (11)? (34) yes no l l yes big only HCCA, DHB (2,7) (11) yes yes no
10 SUMMARY 27 out of 28 labs correctly identified the protein as myoglobin. 24 out of 28 labs correctly identified the Da phosphopeptide. 13 out of 28 labs correctly identified the Da phosphopeptide. Hydrophilicity of the smaller phosphopeptide (741.4 Da) often resulted in loss when fractionated by HPLC. The smaller phosphopeptide was difficult to detect by MALDI-TOF, especially when using HCCA matrix. One lab used neutral loss on a triple quad, but nevertheless missed the smaller phosphopeptide. 7 labs searched for m/z 79 in negative ion mode and all were 100% successful. Remaining labs looked for signals that were multiples of 80 Da (mass of phosphate addition) greater in mass than predicted tryptic peptide molecular weights. QTOF data from 3 out of 28 labs showed best resolution and mass accuracy for main beam (MS1) and product ions (MS2).
11 DISCUSSION & CONCLUSIONS Test samples were requested by 77 laboratories of which 28 returned data sets corresponding to an active participation of 36%. This number is comparable to the participation of 35% in last year's study and is similar to the participation in other ABRF research studies. Suggestions for future studies indicate great interest and the need for studies concerning the identification of proteins and associated problems: participants asked for more complex samples, large molecular weight proteins, limited sample size and the analysis of glycoproteins by mass spectrometry. The time (2-50 hours) laboratories were willing to devote to the analysis of this year's test sample suggests that the sample was treated with as much diligence as any other sample entering core laboratories. Identification of both phosphopeptides depended greatly on the level of experience. With this in mind, it becomes even more difficult to assess all the variables involved in performing these analyses and any comparison of hardware based on such a limited data set should be performed with extreme caution. The overwhelming majority of laboratories were capable of successfully identifying the protein, indicating that the identification of proteins by mass spectrometry represents a reliable methodology. The success rate for correctly identifying both phosphopeptides, large and small, was lower (85% and 46%). The rate of successful complete sequence determination was even lower (75% and 35%). These results indicate that the identification and characterization of phosphorylation sites in proteins using mass spectometric approaches can be a challenging task and still cannot be considered a routine analysis for most laboratories.
12 REFERENCES M. J. Huddleston, R. S. Annan, M. F. Bean and S. A. Carr, J. Am. Soc. Mass Spectrom. 4, (1993). T. Covey, B. Shushan, R. Bonner, W. Schroder and F. Hucho, in "Methods in Protein Sequence Analysis", H. Jorvall, J. O. Hoog, A. M. Gustavsson, Eds.; Birkauser Press; Basel, 1991; pp A. P. Hunter and D. E. Games, Rapid Comm. Mass Spectrom. 8, (1994). ACKNOWLEDGMENTS We wish to thank Dr. Martin Sadilek for blind-coding the data and Dr. Kheng B. Lim for preparing the poster. The poster was prepared using Canvas 5 on a Power Macintosh.
13 KEY & ABBREVIATIONS 1: OWL 2: Swiss Prot 3: PIR 4: nr. fasta 5: Genbank 6: proprietary 7: NCBI nr or nr : EMBL 9: SEQUEST 10: MS-Fit 11: UCSF 12: Sherpa 13: Expasy 14: Prowl 15: ATLAS 16: GPMAW 17: Profound 18: Mass Search 19: ETHZ 20: Peptide Search 21: Patch 22: precursor ion scan in negative mode m/z 79 23: neutral loss scan m/z 49 24: neutral loss scan m/z 98 25: obtained some sequence information for unphosphorylated ASEDLK, confirm phosphorylation by loss of H 3 PO 4 in positive MS/MS mode 26: phosphopeptide identified, but phosphorylation site was not located 27: neutral loss scan (loss of 32.7 u) 28: not clear that the MS/MS spectrum was interpreted to localize Thr 29: study used both MALDI and LC-MS/MS, misidentified 1 phosphopeptide (m/z 1843) looking in negative ion mode by LC-MS/MS 30: according to answer on questionnaire 31: by elimination 32: wrong phosphorylation site 33: guessed incorrectly on 1 phosphopeptide (m/z 1817) 34: used carboxypeptidase on LFTGHPET(p)LEK and confirmed last 4 residues as T(p)LEK, misidentified one phosphopeptide as FKHLKT(p)EAEMK, indicated that protein is not horse heart myoglobin 35: orifice voltage high to monitor m/z 79, low orifice , on-line CE-MS HCCA: alpha-cyano-4-hydroxy-cinnamic acid DHB: 2,5-dihydroxybenzoic acid SA: sinapinic acid
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