MALDI-Tof/Tof Protein Identification

Description of MALDI-Tof/Tof MS Analysis

Protein identification using MALDI-Tof/Tof mass spectrometry is typically performed on 2DE gel spots (e.g., DIGE analysis) or on very pure protein samples after enzymatic digestion. The instrumentation used is the Applied Biosystems (AB) Model 4800 MALDI-Tof/Tof mass spectrometer. After digestion, ~80% of the digest is loaded onto the MALDI target plate using 3mg/ml alpha-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile as the matrix, plus an internal calibrant (1 fmol of bradykinin; protonated, monoisotopic mass = 1060.569) .

Reflectron MS analysis sums 1250 laser shots to generate the peptide fingerprint map (PFM), and the spectra is internally calibrated using the bradykinin internal standard. Masses are chosen by the AB 4000 Series Explorer software (version 3.0) for MS/MS acquisition. An exclude list is used to eliminate the internal standards, and normal trypsin autolysis fragments from MS/MS acquisition. MS/MS can be acquired on 10 to 20 peptides, depending on the amount of sample on the target. Data is analyzed as outlined below.

Protein Identification of DIGE Spots

In a DIGE analysis, the digests are processed as above for MALDI-Tof/Tof analysis. Due to the very low sample amounts for a DIGE analysis of typically ≤ 1 fmol (due to the high sensitivity of the Cy dyes), MS/MS is done first on the masses with the highest intensity, thereby making sure at least a few MS/MS spectra of good quality are obtained before the MALDI spot is depleted. Up to 10 MS/MS spectra are acquired and 10,000 laser shots are combined for each MS/MS spectra. The large number of laser shots is necessary due to the very low sample amounts. This instrument has an extremely high throughput, with MS and MS/MS analysis on 1 sample taking approximately 10 minutes.

Data Analysis

The data is analyzed using the Applied Biosystems GPS Explorer software with Mascot analysis, and a combined peptide mass fingerprinting and MS/MS search. The NCBInr database is routinely searched, with species specific database searches also available. Search parameters include a peptide mass accuracy of 50ppm, a MS/MS fragment accuracy of 0.2Da and variable modifications of cysteine (as appropriate), and methionine oxidation. Mascot search results are loaded into YPED and for analysis of 2DE DIGE spots, the gel image is linked to the protein identification result.

  • Sample Amount=250 to 500 attomols of peptide is routinely identified
  • Recommended amount is 1-10 fmol
  • Coomassie blue and digestion compatible silver stained protein bands are appropriate