Protein Characterization

Molecular Weight Determination

Molecular weight determination for a purified protein or peptide can be performed via LC/ESI/MS using a fast ("ballistic") reversed-phase gradient and a Q-ToF mass spectrometer, or by MALDI-MS. In general, we recommend the Fast LC/ESI/MS because of the inherent sample cleanup that occurs during the workflow to separate your analytes from interfering species that may be in your sample.

Protein identifications based on tandem mass spectrometry

Protein identifications are made based on automated database searching of tandem mass spectra of peptides. This approach was first described in 1994, and, through many refinements, remains state-of-the-art for protein identification.

The Proteomics and Metabolomics Shared Resource uses high resolution (R>10,000) tandem mass spectrometers to provide accurate mass measurements of both peptide molecular weight (MS data) and peptide fragments (MS/MS). High-end mass spectrometers yield high quality data that provide highly confident identification.

Qualitative identifications from 2D PAGE gel spots are performed using either a short (15 min) nanoscale capillary LC gradient coupled to a hybrid Q-ToF mass spectrometer, or using MALDI-MS/MS. Data dependent acquisition and a MASCOT database search or MSE acquisition and Identity software is used to return peptide (and thus protein) identifications.

Qualitative identifications from gel bands or more complex samples, such as cell lysates, are performed using higher-capacity separations (30 min to 2 hours) coupled to Q-ToF mass spectrometers. Higher capacity separation allows for analysis of more complex samples and more confident identification of larger numbers of proteins. Data dependent acquisition and a MASCOT database search, or MSE acquisition and Waters Identity software is used to return peptide (and thus protein) identifications.

For extremely complex samples, two-dimensional separations can be performed prior to MS/MS analysis. The second dimension of these separations is always reversed-phase nanoscale LC, but the first dimensional separation can involve the following mechanisms: strong cation exchange (SCX), strong anion exchange (SAX), hydrophilic interaction chromatography (HILIC), size exclusion chromatography (SEC), or high pH reversed-phase LC. Currently SCX/RPLC and High/Low pH RPLC can be implemented as online LC/LC-MS/MS, while in all other mechanisms, the first and second dimensions of the separation are coupled offline (fraction collection).

Database search engines

Proteomics and Metabolomics Shared Resource uses two principal search engines: MASCOT (Matrix Sciences) and IdentityE (Waters Corporation). Mascot is used for identifications based on serial MS/MS datasets (data dependent acquisition), whereas IdentityE is used for identifications based on multiplexed MS/MS datasets (MSE or Hi-Lo Switching).

1. Identity High Definition Proteomics System

2. Mascot Database

Databases Currently Available

The databases listed below are available via MASCOT or IdentityE for database searching. If your organism of interest is not included in the list here, please check the corresponding websites, or contact the proteomics facility to see if a FASTA format database is available. If so, your favorite organism's database can be added to our search engines for a small one-time setup fee.

1. Swissprot
Mounted Swissprot databases currently include human, mouse, xenopus and E. coli. Other SP databases will require a nominal one-time setup fee.

2. Trembl

Other database resources not currently included, but that can be made available can be found at the National Center for Biotechnology Information (NCBI).

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