Ion Trap LCMS Protein Identification
General Reference
Nature Reviews-Molecular Cell Biology, “The ABC’s (and XYZ’s) of Peptide Sequencing); Steen and Mann, Vol 5, Sept 2004, p699]
We use a Thermo Electron Model LTQ Ion Trap mass spectrometer connected to an Agilent Model 1100 Nanoflow HPLC system. This instrument is excellent for mass spectrometry sequencing (ms/ms) of peptides to identify proteins and has a sensitivity level in the attomole range. Basically, the protein or collection of proteins as in the case of unfractionated pull downs from an immunoprecipitation, are digested with an enzyme, usually trypsin. Different enzymes can be used to increase coverage of the protein. The peptides from the digest are loaded onto a reverse phase capillary separation column and run into the mass spectrometer where they are ionized and their molecular weight determined. The ions are then fragmented in the trap producing mostly type Y and type B ions. We use Sequest software (Anal Chem. 2002 Nov 1;74(21):5593-9. Probability-based validation of protein identifications using a modified SEQUEST algorithm. MacCoss MJ, Wu CC, Yates JR 3rd.) to match the fragmentation patterns with the theoretical fragment patterns of tryptic peptides in the non-redundant protein database from NCBI. Statistical significance of a match is high if one knows the parent molecular weight of a peptide and several contiguous amino acid residues.
We offer three types of services:
- Routine autosampler analysis using capillary column chromatography in which sample is loaded first onto an enrichment trap and then transferred to an analytical C18 column. This is adequate for CBB stained bands.
- Manual loading. This technique has a sensitivity of about 50 femtomoles sample loaded on column.
- Manual loading of individual samples directly onto capillary analytical columns. This technique has a sensitivity of about 5 femtomoles sample loaded on column.
Sample Preparation
Please call the lab or office to discuss your samples if you have not used this service before.
Consider newer fluorescent stains such as Sypro Ruby from Molecular Probes in lieu of silver stain. Sypro ruby appears to give better mass spectrometry results and is easier to use.
We prefer to do the enzymatic digestion ourselves to improve the chance of success. If you perform the digestion, we will insist on seeing your sample prep protocol to avoid damage to our mass spectrometer. No detergents are permissible. Colloidal CBB is very sensitive and works well. If using silver stain, please ask for our hand-out notes regarding keratin contamination elimination and reagent changes to make the stained band more compatible with trypsin digestion. Also see: Gharahdaghi, F., Weinberg, C., Meagher, D., Imai, B., and Mische, S. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601-605 (1999).
Data Output
We email to you the Sequest report in MS Word format. There are many ways to look at the data from a confidence point of view, and you are encouraged to contact the lab to discuss your results.
The following are suggested minimum criteria for data to be considered valid. Each matching peptide fragmentation pattern should be inspected for accuracy. Two or more different peptide matches inspire confidence, although it is possible to identify a protein from a single match (a.k.a. one-hit-wonders) after careful inspection of the fragment pattern match from a fairly long peptide and after making sure that pI, MW, species, function, etc. all make sense.
Charge State |
Xcorr |
Sp |
Delta CN |
|---|---|---|---|
+1 |
>1.5 acceptable, >2.0 good, >1.9 rigorous |
>750 |
>0.1 |
+2 |
2.0 acceptable, >2.2 good, >2.7 rigorous |
>750 |
>0.1 |
+3 |
>2.5 acceptable, >2.9 good |
>750 |
>0.1 |
