MASS SPECTROMETRY & PROTEOMICS
LAB COMPUTER EXERCISES INTRODUCTION


MASS SPECTROMETERS

COMPUTER EXERCISE #1: Observe how one type of mass spectrometer works.

Select this link to observe how an instrument works
Electrospray source, Ion Trap analyzer:
LCQ and ESI Ion Trap System

INFORMATION ABOUT PROTEINS

COMPUTER EXERCISE #2: Find the theoretical MW of a known protein.  Compare theoretical to observed.  Browse information and view structure.

(a)  Open an internet browser, go to the Expasy Proteomics Server:  http://us.expasy.org/

Select SWISS-PROT and TrEMBL database

Type: alkaline phosphatase ecoli
Will result in information about protein precursor (P00634)
Explore the information (Feature Table, amino acid sequence)

Select Computer pI/Mw
Select residues 22-471 (this is the protein without signal)
This will provide information about the protein's average MS

Compare the mass spectrum (below) to the theoretical average MW.  Calculate the m/z difference and % error between the observed and theoretical protein.


Figure 1.  MALDI-TOF spectrum of alkaline phosphatase

(b) To view the structure of this protein, open a new browser page, go to the Protein Data Bank http://www.rcsb.org/pdb/

Search the Archive for Alkaline phosphatase e.coli, type in the ID number, do a full text search: (P00634)
Select Search
Select any of the structures listed
Under DISPLAY OPTIONS select a viewer
Explore the information provided by this page
Try other proteins, use the "search" option on the left side of the main page, select browse database and select any section, then any protein.

2-D GEL ELECTROPHORESIS

COMPUTER EXERCISE #3: Explore information on a 2-Dimensional gel.

Go to http://us.expasy.org/ and select SWISS-2DPAGE (under databases).
Choose access to Swiss 2D page by assession number
Search for P00359 (Glyceraldehyde 3-phosphate dehydrogenase from yeast)
Enlarge the gel and notice where the spot is observed.

Return to SWISS-2DPAGE, and choose access by clicking on a spot .
Find the gel for yeast (Saccharomyces cerevisiae) and try to find the same protein.


PROTEIN IDENTIFICATION BY PEPTIDE MASS MAPPING

COMPUTER EXERCISE #4: Investigate peptide mass mapping used for protein identification



Figure 2.  MALDI-TOF spectrum of tryptic peptides from Unknown protein #116.

(a) Identify a protein from measured peptide masses

Open the excel spreadsheet provided in the link excel data .  Use only the tabs labelled 66, 166, 55, 36.
Copy and paste the m/z values into the search described below.
Delete the m/z values 904.4681 and 2465.199 (these are internal calibrants)

Identify unknown protein #66 using the excel data.
Identify protein from other unknown proteins (#166, 55, 36) as time permits.

Open an internet browser, go to http://prospector.ucsf.edu/
Select MS-Fit program

At the bottom of the screen is a data paste area filled with masses.
Delete these and copy/paste the mass list from the excel spreadsheet.
Search these masses while changing different options.
Options to vary:  (Database (swiss-Prot, Owl, NCB), Mass tolerance (50, 70, 100, 120 ppm)
Look for proteins with strong MOWSE scores with low mass errors.

(b) Find a protein's theoretical peptides to confirm the identity

Open an internet browser, go to http://prospector.ucsf.edu/

Select the MS-Digest program

Options to choose:
Retrieve entry by assession number (Enter a number for the protein identified in #3a)
Select the database where your protein was found
The digestion enzyme is Trypsin, with 0 missed cleavages

BLAST SEARCHING

COMPUTER EXERCISE #5: Investigate the use of unannotated sequence data contained in databases of genome sequencing information. Perform BLAST searching to find homologous proteins.
A protein was subjected to nanoLC-MS/MS and two peptides were identified by Sequest searching against a database of rice genome sequence data (which is not publicly available). The protein header is listed simply as “unknown”. See tab in exercise spreadsheet for details of the peptide sequences.  Perform a BLAST search to find similar proteins, and get some idea what the function might be of this unknown protein..

Open a browser, go to www.ncbi.nlm.nih.gov/BLAST
Choose the protein-protein (Blastp) option

Copy and paste the two peptides sequences into the “search” window as a single string of text i.e. Make sure to remove the dots and any unnecessary spaces.
Check that the database searched is set to nr (i.e. the nonredundant database)

When the new page appears that indicates you search has been submitted, press the format button. Your results (when ready) will open in a new window)
Look at the results, ignoring the “unknown” proteins and see if there is a common functional theme among the various proteins reported as having decent homology.  (Not necessarily the first one.)  Look at some of the individual matches and see how closely (or not) they resemble the sequence you input.

PROTEIN IDENTIFICATION BY MS/MS

COMPUTER EXERCISE #6: Investigate the use of MS-MS data for peptide sequence information -- and ultimately protein identification.

An unknown protein was digested with trypsin and MS-MS spectra was acquired.
A list of fragments from one of these peptides is included in the excel spreadsheet (fragments 316-318).

Go to Protein Prospector , select the MS-Tag program.
Copy/paste the mass list, overwriting the default masses on the program.
First on the list should be the mass of the selected peptide (this may need to be calculated)

Search for possible peptides/proteins.

MORE PROTEINS TO IDENTIFY (MS/MS)

COMPUTER EXERCISE #7: Hard copies of MS/MS spectra will be provided.  

    Identify the amino acid sequence of a peptide using the method shown in #5.  The results will identify the protein .


X TANDEM (MS/MS)

COMPUTER EXERCISE #8: Raw data files in electronic form of MS/MS spectra will be provided.

Identify proteins using the XTandem program found at www.theGPM.org Click on "Genomes" to go to data entry page.

The program will use data from *.dta files that can be downloaded and run on the server at the GPM.

luci_1141.dta = a single MS/MS spectrum from a run

Luci_combined.dta = all MS/MS spectra from the same run

Ecoli_01_combined.dta = 1D gel band

Ecoli_05_combined.dta = 1D gel band

Ecoli_08_combined.dta = 1D gel band

Ecoli_09_combined.dta = 1D gel band

Parameters: Select database (e.coli), modifications (carbamidomethyl, oxidation of M), enzyme (trypsin), predefined methods (ion trap), add to GPMDB (no).

Output: view protein for some of the results, explore homologues, and other links