Peptide Mass Fingerprinting by MS-MALDI TOF

Service description

The more efficient approach for rapid identification of isolated proteins from sequenced organisms. The analysis can be carried starting from a liquid solution of the target protein or from protein bands separated by gel electrophoresis. In the last case, the gel spots/bands with the target proteins are first cut and then the protein in the gel slice digested in situ with a specific enzyme, generally trypsin. The digestion peptides are extracted and analyzed consecutively by MALDI TOF MS. MALDI TOF MS A fraction of the peptide digest is placed on a MALDI plate where it is mixed with the MALDI matrix (i.e. alpha-ciano-4-hidroxicinamic acid) and it is let to dry. The crystalized sample is analyzed in a MALDI-TOF (4800 TOF/TOF, ABSciex) mass spectrometer using the positive ion reflector mode. The analysis is made in automatic mode averaging the spectra obtained from 4000-5000 shots in the m/z range from 750 to 4500. Each spectrum is calibrated externally by using a standard 5-peptide mix (des-Arg1-bradykinin (Mr 904.46), Glu1-fibrinopeptide B (Mr 1570.68), angiotensin-1, (Mr 1296.69), ACTH 1-17 (Mr 2093.09), ACTH 18-39 (Mr 2465.20), ACTH 7-38 (Mr 3657.93) and, when possible, an internal calibration using the ions derived from the trypsin auto-digestion (Mr 842.5100, 1045.5642, 2011.1046, 2807.3145 and 3337.7577). The spectra obtained contain the accurate mass of the digestion peptides present in the sample. This set of peptide masses constitutes a fingerprint of the original protein sequence. Proteins are identified from these peptide mass fingerprints (PMF) using bioinformatic tools that match them with those expected from the sequences in protein databases. Note: Whenever the protein cannot be identified via PMF or confirmation of the PMF data is required, a TOF/TOF MS/MS analysis can be carried over the same sample (See PEPTIDE MASS FINGERPRINTING MS - MS/MS BY MALDI TOF/TOF). Alternatively, the protein digests can be analyzed by electrospray MS/MS (See LC MS/MS identification and nESI MS/MS identification). This approach is complementary to MALDI-TOF/TOF, providing of a different ionization selectivity and often of a more efficient sequenciation procedure. ESI MS/MS is however more time consuming and have a higher economic cost, so that for routine identification and/or identification of high numbers of protein samples, you still want to try MALDI TOF/TOF first.

Service description

Peptide mass fingerprinting (PMF) is an analytical technique for protein identification. Basically, the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with MALDI-TOF mass spectrometer. Then these masses are compared to a database containing known protein sequences. Then the absolute masses of the peptides from each protein are calculated theoretically for mass comparison between the peptides of the unknown protein and the theoretical peptide masses of each protein to find the best match. In sample preparation for PMF, protein samples can be derived from SDS-PAGE or in protein solution and then subject to some chemical modifications. Then the proteins are cut into several fragments using proteolytic enzymes to generate peptides for mass spectrometric analysis. A small fraction of the peptide is pipetted onto a MALDI target with a suitable matrix and then the matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam. The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal. The mass spectrometric analysis produces a list of molecular weights of the fragments. The peptide masses are compared to protein databases such as Swissprot, NCBInr, which contain protein sequence information. The results are statistically analyzed and possible matches are returned. The mass spectrometer that we use for the analysis is an Autoflex III MALDI-TOF/TOF. A suite of software is used with this instrument. Acquisition of mass spectra is done in Flex Control, spectrum processing and annotation in Flex Analysis, and additional data processing and data searching is done in BioTools and Sequence Editor (Bruker). The search engine used for the protein identification is MASCOT (MatrixScience, UK).