It is reasonable to perform internal or external calibration with a test-series of protein fragments having known masses, such as bombesin , substance P or the adrenocorticotropic hormone ACTH 18-39 clip . There are also internal calibration methods where the autolytic peptides of the enzyme used for digestion are selected, for example the trypsin fragments at m/z 842.50, 1045.56 and 2211.10, which can yield a mass accuracy up to 20 ppm (Friedman et al. 2004).

A database search with the peptide mass fingerprint (PMF) of the analyte can be performed against different databases like:

MSDB ( www.dbarecovery.com/restoremsdb.html )

NCBInr (http://www.ncbi.nlm.nih.gov/),

PIR (http://pir.georgetown.edu/)

SwissProt (http://us.expasy.org/sprot/)

using different search engines like MasCot (www.matrixscience.com), Prospector or ProFound (http://prowl.rockefeller.edu).

With these different search engines, very convenient automation steps are possible.

The validation procedures of the gained PMFs differ between search engines depending on the internal algorithms used. The parameter settings should be based on the taxonomy of the sample species, the used enzyme for hydrolysis and the mass accuracy. It is also not advisable to allow more than one missed cleavage. It is recommended that each PMF analysis should be based on at least two separate digests from the same protein picked from two different gels. The most concise criterion for final protein identification in the data-base search is its score, with the level of significance for the match.

Peptide sequence. It can be performed by:

• PSD
• sequencing of the peptides via classical Edman degradation

Both methods, however, require much higher protein amounts than PMF in order to obtain reliable results. A Western-blot of a second 2D-gel with an available antibody of good selectivity for the identified protein may also be performed. Blotted gels may also be exposed to commercially available antibody cocktails with different labelling and detection procedures. Customized libraries of antibodies, or sets of antibodies for specific pathways or processes such as cancer- or apoptosis-relevant proteins can be purchased.

Recently, in vivo stable isotope labelling of proteins has been combined with 1D- and 2D-gelelectrophoretic separation followed by MALDI-TOF MS peptide fingerprinting. This approach reveals the identity of individual proteins, but at the same time from the relative rate of incorporation of the label also informations about the metabolic turnover rate can be abstracted from the spectrum. Applied to mature 3T3-L1 adipocytes a high rate of remodelling of extracellular matrix proteins and in particular of the type-I collagen fibers was demonstrated (Bouwman et al. 2004).