**7. Protein identication and validation**

There are a number of different methods for identifying the proteins in the sample, and the most frequently used is the searching of the uninterpreted MS/MS data. The FASTA formatted protein sequences from National Center for BioTechnology Information (NCBI) and UniProtKB/Swiss-Prot databases are collected for proteins identied or identification by each MS experiment. Searching uninterpreted MS/MS data from a single peptide or from a complete nanoLC-MS/MS run was automatically analyzed with a non-redundant protein database by the program SEQUEST, which allows the correlation of experimental data with theoretical spectra generated from known protein sequences [7].

2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins 73

**Figure 5.** Illustration of 2D-nanoLC-ESI-MS/MS spectra: (a) The total ion current (TIC) profile tryptic digest of membrane proteins (band 6) at the concentration of 100 mM ammonium acetate for run time 0- 50 min; (b) TOF-MS spectrum at 16.054 min; (c) TOF product spectrum of a peptide ion with m/z = 510.45.

(c)

(a)

(b)

The precursor mass is used as a lter to nd a list of candidate peptide sequences from the theoretical digest of the database. A variety of different systems are used to score the experimental MS/MS spectrum against spectra predicted from the candidate peptide sequences. For protein identication, experimental data were searched against the NCBInr and Swiss-Prot mouse protein database using Mascot v1.8 software in which the criteria were based on the manufacturer's denitions (Matrix Science Ltd, London, UK) [20]. The parameters were set as follows: enzymatic cleavage with trypsin; 1 potential missed cleavage; a peptide and fragment mass tolerance of ± 0.25 Da, and xed modication of carbamidomethyl (cysteine); variable modication of oxidation (methionine); 1+, 2+, and 3+ peptide charge. Protein identications were performed using a Mowse scoring algorithm with a condence level of 95% and at least two matched peptides, showing a high score [12].

For further verication, proteins might be validated by MSQuant software [1, 4, 24] available at http://msquant.sourceforge.net. The MSQuant software is used as a validation and quantitation tool that produces the Mascot peptide identications (HTLM les) and allows manual verication against the raw MS data (QSTAR XL raw les). The MSQuant software will pick up signicant and veried hits from the Mascot output le and export information of identied proteins into an .xls le, including the GI (genInfo identifier) number and molecular-mass values.
