UC Mass Spectrometry Facility & Protein

Characterization for Proteomics Core

Proteomics Capabilities: Examples of Protein ID and

Analysis of Modified Proteins to Establish Protein-Protein

Interactions

Excised gel spots are submitted to MSF for analysis. After proteolysis, liquid chromatography tandem mass spectrometry (LC-MS/MS) is done.

Expanded view of spots 1 & 2

1 2

35

46

7

2-D gel with spots to be analyzed

Protein Identification

Protein IdentificationAfter LC-MS/MS, the data file is searched against a protein database. Significant protein matches (outside the green shaded box) are identified based on the quality of the peptide sequences obtained by LC-MS/MS. The list of significant matches are then reported back to the investigator.

Protein Identification

Facility personnel verify protein assignments by examining quality of underlying sequence data. Primary data is also archived and can be re-examined upon investigator request.

Protein Identification

For Protein Identification of gel spots:More sample submitted yields higher quality

resultsSome samples may contain mixtures of proteins

Protein Identification

In addition to protein identification from gel spots, mixtures of proteins (e.g., pulldowns) can be analyzed in asimilar fashion. In this case, a combination of data reduction approaches are used to generate protein identifications.

Initial level - database searchSecond level - de novo sequencing and BLAST

searchFor this example, nearly 30 unique proteins were identified from an investigator supplied protein mixture.

Case study #1: Protein-Protein interactions among three extrinsic membrane proteins

Modified ProteinsThe mass spec process for characterizing protein modifications is significantly different than that used for protein identification.

There are three keys to success:

1. Availability of pure control and modified protein in reasonable abundance

2. Availability of control protein sequence

3. High sequence coverage in the MS analysis

The first step in the process is to obtain as much MS and MS/MS information from the control and modified protein as possible.

Modified Proteins

TIC MS Scan

TIC MS/MS Scan

ModifiedControl

TIC MS Scan

TIC MS/MS Scan

Modified Proteins

Among the approaches for increasing sequencing coverage are the use of multiple proteases and/or variation in the MS experimental conditions. When completed, nearly 350 peptide sequences needed to be evaluated for this 17 kDa protein.

1 EARPIVVGPPPPLSGGLPGTENSDQ26 ARDGTLPYTKDRFYLQPLPPTEAAQ51 RAKVSASEILNVKQFIDRKAWPSLQ76 NDLRLRASYLRYDLKTVISAKPKDE101 KKSLQELTSKLFSSIDNLDHAAKIK126 SPTEAEKYYGQTVSNINEVLAKLG

17 kDa extrinsic membrane protein

Modified Proteins

Next, a database of potential modified peptides is generated to be used for searching of experimental data

ModificationsStart End Sequence singly chargeddoubly charged1LCBt 28 37 (R)DGTLPYTKDR(F) 1391.6629 696.335351LCBt 28 51 (R)DGTLPYTKDRFYLQPLPPTEAAQR(A) 3003.5038 1502.25581LCBt 38 63 (R)FYLQPLPPTEAAQRAKVSASEILNVK(Q) 3096.6556 1548.83171LCBt 52 63 (R)AKVSASEILNVK(Q) 1484.8146 742.91121LCBt 52 68 (R)AKVSASEILNVKQFIDR(K) 2144.1537 1072.580752LCBt 52 68 (R)AKVSASEILNVKQFIDR(K) 2370.2313 1185.619551LCBt 54 68 (K)VSASEILNVKQFIDR(K) 1945.0217 973.014751LCBt 54 69 (K)VSASEILNVKQFIDRK(A) 2073.1166 1037.06221LCBt 64 79 (K)QFIDRKAWPSLQNDLR(L) 2213.1289 1107.068351LCBt 82 98 (R)ASYLRYDLKTVISAKPK(D) 2179.1949 1090.101352LCBt 82 98 (R)ASYLRYDLKTVISAKPK(D) 2405.2724 1203.14011LCBt 87 98 (R)YDLKTVISAKPK(D) 1588.8772 794.94252LCBt 87 98 (R)YDLKTVISAKPK(D) 1814.9548 907.98131LCBt 87 101(R)YDLKTVISAKPKDEK(K) 1961.0417 981.024752LCBt 87 101(R)YDLKTVISAKPKDEK(K) 2187.1193 1094.063553LCBt 87 101(R)YDLKTVISAKPKDEK(K) 2413.1969 1207.102351LCBt 91 98 (K)TVISAKPK(D) 1069.6079 535.307851LCBt 91 101(K)TVISAKPKDEK(K) 1441.7724 721.39011LCBt 91 102(K)TVISAKPKDEKK(S) 1569.8674 785.43762LCBt 91 101(K)TVISAKPKDEK(K) 1667.85 834.42892LCBt 91 102(K)TVISAKPKDEKK(S) 1795.945 898.47643LCBt 91 102(K)TVISAKPKDEKK(S) 2022.0225 1011.515151LCBt 99 102(K)DEKK(S) 745.3554 373.18161LCBt 99 110(K)DEKKSLQELTSK(L) 1631.8314 816.41962LCBt 99 110(K)DEKKSLQELTSK(L) 1857.909 929.45841LCBt 102 110(K)KSLQELTSK(L) 1259.6669 630.337351LCBt 102 123(K)KSLQELTSKLFSSIDNLDHAAK(I) 2671.3765 1336.192152LCBt 102 123(K)KSLQELTSKLFSSIDNLDHAAK(I) 2897.4541 1449.23095

Modified Proteins

A list of potentially modified peptides is generated after the database search; initial evaluation of likelihood of modification is then made (similar to MALDI-based MS methods).

1 EARPIVVGPPPPLSGGLPGTENSDQ26 ARDGTLPYTKDRFYLQPLPPTEAAQ51 RAKVSASEILNVKQFIDRKAWPSLQ76 NDLRLRASYLRYDLKTVISAKPKDE101 KKSLQELTSKLFSSIDNLDHAAKIK126 SPTEAEKYYGQTVSNINEVLAKLG

Mods? Start End Sequence Singly ChargedDoubly ChargedExp m/z1 Biotin 91 102(K)TVISAKPKDEKK(S) 1569.8674 785.4376 785.40621 Biotin 99 110(K)DEKKSLQELTSK(L) 1631.8314 816.4196 816.42552 Biotin 91 102(K)TVISAKPKDEKK(S) 1795.9450 898.4764 898.45201 Biotin 54 68 (K)VSASEILNVKQFIDR(K) 1945.0217 973.0148 973.01292 Biotin 52 68 (R)AKVSASEILNVKQFIDR(K) 2370.2313 1185.6196 1185.5529

Modified Proteins

All potentially modified peptides are then de novo sequenced to determine whether modification site can be seen from the sequence data.

Modified Proteins

Several software programs are used for de novo sequencing. For the 973.01 ion, a biotinylated residue at K63 was found. Site of modification is determined by the 226 Da mass shift of the y6 peptide fragment ion.

Query – 973.01,+2Sequence determined – (54)VSASEILNVKQFIDR(68)

K + biotin

Modified Proteins

Although the initial mapping data may suggest possibility of modification, in some cases insufficient sequence information is available to confirm possibility. Analysis of other LC-MS/MS runs is then required.

Query – 785.42,+2No sequence determined

Modified ProteinsCharacterization of Modified Proteins:

1. LC-MS/MS of control sampleOptimize chromatographic & mass spectrometric

conditionsDetermine sequence coverageEstablish protein database (when necessary)

2. Generate database of potential modification sites3. LC-MS/MS of modified sample

Compare LC-MS information to generated database values; identify potential modified peptides

De novo sequencing of potential modified peptides Verify assignments or retrieve additional information

with other proteolytic digestions

Modified ProteinsMS Facility Helpful Hints:

Purify at least 1 µg control and 1 µg modified protein

Be specific about the information desired (e.g., number of modification sites; specific sites of modification; possibility of undermodification)

Be patient – characterization of modified proteins is not a short turn-around time analysis

Contact Dr. Macha (6-1575) beforehand for guidance and facility backlog information