Protein Identification - Technical Details
Samples
MALDI peptide mass fingerprinting can be performed from 1D&2D-gel plugs after fluorescence staining (Cy-Dyes, Sypro-Ruby and similar), MS-compatible silver-stain, Coomassie G250 and R250 stain. MALDI peptide mass fingerprinting from dryed gels is possible, however, samples from fresh wet gels reveal significantly better protein identification results.
2D-gel plugs stained with silver (left), Coomassie G250 (middle) and Coomassie R250 (right). PCE-MM200 Digital USB Microscope (200x).
(Courtesy of WITA GmbH, Teltow, Germany)
Please observe the following recommendations:
In general: Avoid to expose your samples to protein modifying conditions. Please consult Proteome Consult concering the compatibility of your sample preparation procedures with respect to MALDI peptide mass fingerprinting.
1D SDS-PAGE-samples: Do not boil or heat your samples. Exposing your 1D-samples to 95°C will complicate the identification of proteins via MALDI peptide mass fingerprinting.
2D-gel samples: Do not heat the samples. Do use ultrapure urea. Avoid thiourea.
Tryptic Digest
Samples are digested using sequence grade modified trypsin (Roche Diagnostics GmbH, Penzberg, Germany) according to Proteome Consults C18-free protocol.
Sample Preparation
Digests and extracts are spotted onto Bruker AnchorChipTM 400/384er targets using a-Cyano-4-hydroxycinnamic acid (HCCA) as matrix.
12 'real life' HCCA target preparations (left) and clean anchor (right). Bruker AnchorChipTM 400/384er target. PCE-MM200 Digital USB Microscope.
(Courtesy of WITA GmbH, Teltow, Germany)
Sensitivity
The sensitivity of the method has been tested using an equimolar mixture of 5 synthetic peptides of pI-values ranging from pI=4.4 to pI=12.3 and masses within the peptide mass finger print range. Peak annotation was performed by Bruker Flexanalysis 2.0 software using the SNAP-algorithm.
MALDI-MS spectra of the 5 synthetic peptides at 2nmol. HCCA-Matrix, Bruker AnchorChipTM 400/384er target.
MALDI-MS spectra of an equimolar mixture of 5 synthetic peptides within the low femto-mol to high atto-mole range.HCCA-Matrix, Bruker AnchorChipTM 400/384er target.
(Courtesy of WITA GmbH, Teltow, Germany)
The results demonstrate low femto-mole to high atto-mole sensitivity for all peptides under routine conditions used for protein identification via MALDI peptide mass fingerprinting.
Database Searching
Due to the high sensitivity of the method two or more proteins may be identified from 2D-gel spots and especially from 1D-gel bands. Therefore high resolution separation techniques for sample preparation are required for complex protein mixtures like cell or tissue extracts.
Mascot protein identification (positive control) of 2D-gel spot obtained from Sigma A5503 Albumin chicken egg, grade V, purity >98%.
(Courtesy of WITA GmbH, Teltow, Germany)
Database searching is performed within a local copy of the NCBInr-database. Customer provided databases in FASTA-format may be incorporated on request.
SYPRO® is a registered Trademark of Molecular Probes Inc., Eugene (USA)
AnchorChipTM is a Trademark of Bruker Daltonics, Bremen (Germany)
