Thermo Fisher Scientific

Protein analysis and identification via mass spectrometry is performed by either the Top-Down or Bottom-Up approach.   The most common method to identify proteins in mixtures or from gel spots is the Bottom-Up approach. With this technique, even very complex samples are digested with highly specific cleavage agents such as Trypsin. The resulting peptide mixtures are often extremely complex and require further separation by 1D or 2D HPLC prior to mass spectrometric analysis. The digestion of the sample prior to on-line HPLC-MS measurement is time consuming but necessary for breaking the protein into smaller peptides which allow the protein identification by searching their MS/MS product ion spectra against protein databases.

Analysis of intact proteins by the Top-Down approach avoids digestion, additional separation steps, and sample loss, although desalting is necessary in many cases.

The requirements for such analysis are high resolution at fast scan repetition rate in combination with high mass accuracy. This is provided by the LTQ FT, which is the combination of a linear ion trap and an ICR analyzer.

Using electrospray ionization on the LTQ FT analysis of intact proteins result in multiply charged molecular ions (see figure 1) observed in the normal scan range of the instrument (m/z 400-4000 ). Multiply charged species can be isolated in the linear ion trap and fragmented in a Data Dependant™ manner in both the ion trap part (CID ), or in the ICR cell using ECD or IRMPD, or even in the simultaneous combination of both. Also the ions can be detected with either the ion trap detection system or with the ICR cell detector utilizing high resolution and high mass accuracy.

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Figure 2 shows a representative fragmentation spectrum containing different series of multiply charged fragment ions (top trace) which is simplified by deconvolution to the singly-charged mass spectrum making ion assignment easier (middle and bottom traces). This step is done with Thermo’s Xtract™ software that determines the charge state of each ion species by calculating the mass distance of adjacent isotope peaks resulting in either singly or ‘zero charged’ spectra. The generated peak list of the monoisotopic fragment ions allows final database searching.

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Data interpretation is performed with the Protein Calculator (Thermo Scientific software). This program allows protein sequence input from any ‘fasta’ database, modify and/or digest the proteins in silico, generate MS/MS fragments of all known ion series, and compare those to the measured data. Another tool is the web based ‘Prosight’ search engine (Univ. Illinois) which is accessible at https://prosightptm.scs.uiuc.edu.

Due to its high resolving power and excellent mass accuracy, the Thermo Scientific LTQ FT is the ideal tool for Top-Down analysis for off-line and on-line measurement of intact proteins.