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Proteomics Capabilities: Examples of Protein ID and Analysis of Modified Proteins to Establish Protein-Protein Interactions. UC Mass Spectrometry Facility & Protein Characterization for Proteomics Core. Protein Identification. 2-D gel with spots to be analyzed. 1. 2. 3. 4. 6. 5. 7.

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protein identification
Protein Identification

2-D gel with spots to be analyzed

1

2

3

4

6

5

7

Expanded view of spots 1 & 2

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

protein identification1
Protein Identification

After 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 identification2
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 identification3
Protein Identification

For Protein Identification of gel spots: More sample submitted yields higher quality results Some samples may contain mixtures of proteins

protein identification4
Protein Identification

In addition to protein identification from gel spots, mixtures of proteins (e.g., pulldowns) can be analyzed in a

similar fashion. In this case, a combination of data reduction approaches are used to generate protein identifications.

Initial level - database search

Second level - de novo sequencing and BLAST search

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

modified proteins
Modified Proteins

The 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

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

modified proteins1
Modified Proteins

Modified

Control

TIC MS/MS Scan

TIC MS/MS Scan

TIC MS Scan

TIC MS Scan

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 proteins2
Modified Proteins

17 kDa extrinsic membrane protein

1 EARPIVVGPPPPLSGGLPGTENSDQ

ARDGTLPYTKDRFYLQPLPPTEAAQ

51 RAKVSASEILNVKQFIDRKAWPSLQ

76 NDLRLRASYLRYDLKTVISAKPKDE

101 KKSLQELTSKLFSSIDNLDHAAKIK

126 SPTEAEKYYGQTVSNINEVLAKLG

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.

modified proteins3
Modified Proteins

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

modified proteins4
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 EARPIVVGPPPPLSGGLPGTENSDQ

ARDGTLPYTKDRFYLQPLPPTEAAQ

51 RAKVSASEILNVKQFIDRKAWPSLQ

76 NDLRLRASYLRYDLKTVISAKPKDE

101 KKSLQELTSKLFSSIDNLDHAAKIK

126 SPTEAEKYYGQTVSNINEVLAKLG

modified proteins5
Modified Proteins

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

modified proteins6
Modified Proteins

K + biotin

Query – 973.01,+2

Sequence determined – (54)VSASEILNVKQFIDR(68)

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.

modified proteins7
Modified Proteins

Query – 785.42,+2

No sequence determined

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.

modified proteins8
Modified Proteins

Characterization of Modified Proteins:

1. LC-MS/MS of control sample

Optimize chromatographic & mass spectrometric conditionsDetermine sequence coverageEstablish protein database (when necessary)

2. Generate database of potential modification sites

3. 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 proteins9
Modified Proteins

MS 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