Protein Identification by Mass Spectrometry

Protein samples are commonly submitted as stained gel bands which are then digested directly in the gel by a proteolytic enzyme, usually trypsin. Samples in solution may also be submitted for in-solution digestion, which may be desirable when greater sequence coverage is needed, e.g. identification of post-translational modifications. Trypsin is the enzyme of choice for its specificity and reliability, and also because resulting peptides contain C-terminal Lys or Arg residues, making them especially amenable to ionization in the mass spectrometer. Other enzymes, such as endoproteinase Lys-C, chymotrypsin, and subtilisin, are also used when an alternative to trypsin cleavage is required.

Two mass spectrometry methods are available for identifying the digested proteins: MALDI-TOF and ESI-LC-MS/MS:

MALDI-TOF: MALDI-TOF(matrix-assisted laser desorption ionization time-of-flight) mass fingerprinting, also called peptide mass mapping, uses the analyzed masses of the tryptic peptides to identify a protein by comparison to the calculated masses of tryptic peptides of all proteins in the current non-redundant database. This is done by submitting experimental data to one of several open access programs on the web:

• http://prowl.rockefeller.edu/prowl-cgi/profound.exe
• http://prospector.ucsf.edu/prospector/4.0.7/html/msfit.htm
• http://www.matrixscience.com/search_form_select.html

Hits are scored and ranked, and the top hit is verified by Core staff based on sequence coverage, mass error, and presence of expected modifications.

Note that mass fingerprinting does not give sequence information; only peptide masses are measured. MALDI works well for 2D gel spots and 1D bands containing predominantly one protein, e.g. verification of the identity of an expressed protein. It is not the preferred method for more complex 1D bands, e.g. identification of proteins from a pulldown experiment. MALDI cannot be used for organisms for which the genome is incomplete, as it relies on matching the experimental data to proteins in the public databases.

ESI-LC-MS/MS: Nanoflow liquid chromatography is coupled with electrospray ionization to analyze peptides in complex mixtures, such as an enzymatic digest. Two mass measurements are performed: the mass of the intact peptide as it elutes from the column and enters the mass spectrometer, and the masses of the fragments produced when the peptide undergoes collision-induced dissociation (CID), hence the term tandem mass spectrometry or MS/MS. Because peptides have predictable fragmentation patterns based on their sequence, the experimental data from each peptide can be searched against the predicted fragment ions for all peptides in the available database, and the protein from which it originates can be identified. This is done through a program such as Mascot: http://www.matrixscience.com/search_form_select.html

Mascot groups multiple peptides from the same protein together and assigns a score. In this way, up to 20 protein components in a single sample can be identified, making this method suitable for 1D gel bands from pulldown or IP experiments in which more than one protein is likely to be present.

Unlike MALDI spectra, MS/MS data from an ES-LC-MS/MS experiment give information about the sequence of individual peptides, and is used to verify post-translational modifications or sequence variations. It can also be used for de novo sequencing in which the MS/MS spectrum is manually interpreted to determine a stretch of sequence which can then be used in a homology search or to construct a oligonucleotide probe. De novo sequencing is frequently used in the Core for incomplete genomes. Success of this method is dependent on the quality of MS/MS data which is in turn strongly dependent on the amount of protein in the original sample.

Instrumentation

The Mass Spectrometry Core has an Applied Biosystems Voyager DE Pro purchased in 2005 for MALDI analysis.

LC-MS/MS is routinely performed on a Micromass QTof I equipped with an LC Packings nanoflow LC. Typically 5 µl of digest solution is injected onto an LC Packings C18 PepMap column (0.75 µm x 15 cm) and eluted with a linear acetonitrile gradient at a flow rate of 200nL/min. The peptides eluting from the column are introduced into the mass spectrometer through a New Objective PicoTip held by a New Objective adapter. The Mass Spectrometry Core also has an Applied Biosystems QSTAR XL coupled to an Eksigent nanoflow LC with a 10-port valve which can be used for on-line 2D chromatography (ion exchange followed by reverse-phase).

| TOP |