Service description
The more effcient 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, fthe 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 and MALDI TOF/TOF MS/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 ABSCIEX 4800 TOF/TOF (ABSciex, Biosystems, Barcelona) 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.
MALDI TOF/TOF MS/MS
Sometimes, when only a few digestion peptides are observed for a protein or when there are interferences in the MALDI spectra from other contaminating proteins, the PMF analysis can fail, produce ambiguous results or results of low confidence. For highly confident identification, the PMF analysis can be followed by an MS/MS analysis where several of the most abundant ions detected in the PMF spectra are automatically selected and fragmented (MS/MS) to obtain additional sequence data for unambigous identification of the protein.
Note:
Whenever the protein cannot be identified via PMF or PMF-MS/MS, 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 efficent 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 proteins, you still want to try MALDI TOF/TOF first.
Options and prices chart
Options |
Unit |
Public Sector |
Other customers |
PMF y PFF (Fragmentación peptídica) |
€ / muestra
|
49.41 € |
54.11 € |
PMF |
€ / muestra
|
37.56 € |
41.13 € |
Caracterización de proteínas con MALDI-TOF/TOF |
€ / muestra
|
74.1 € |
81.16 € |