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Protein Identification

Electrospray Mass Spectrometry

        • Protein/peptide identification from enzymatic digest
        • Identifiction of protein modifications
        • Small molecules/metabolites analysis
        • $40 per run for LTQ, $30.00 per run for DECA (for specific pricing contact the Core Facility)
        • Please contact the facility if you are interested in this service at (806) 742-2393.
        • Instrument details listed below
        • Electrospray Mass Spectrometry Order Form

 

MALDI-TOF/TOF Mass Spectrometry

 

Electrospray Mass Spectrometer Instruments Detail

The LTQ-XL mass spectrometer is an electrospray system coupled with a Dionex nano-HPLC (Ultimate 3000) system. The instrument is used for protein/peptide identification from an enzymatic digest. A protocol of in-gel tryptic digest of a protein band is available on our website. Peptides separation is performed on a Dionex nano-HPLC (Ultimate 3000) with a trapping column (C18, 3 μm, 100 Å, 75 μm × 2 cm) followed by a reverse phase column (C18, 2 μm, 100 Å, 75 μm × 15 cm, nanoViper). Peptides are first injected onto the trapping column and washed for 10 min and then the trapping column is switched to the reverse-phase analytical column and bound peptides eluted using solvents A (2% ACN, 0.1% formic acid in water) and B (98% ACN, 2% water, 0.1% formic acid). The eluted peptides are separated on a C18 column and directed into the nanospray ionization source of the LTQ-XL.

Using Proteome Discoverer software, the MS and MS/MS data are used to search against databases for identification and often detect the exact sequence location of modifications on the peptide (eg: phosphorylation sites).
Further Quantitative analysis of proteome in the mixture can also be performed using our new Proteo IQ 2.6 software based on spectral counting technique.

The Thermo Electron DECA XP+ electrospray ionization mass spectrometer (ESI MS) in line with a Dionix UHPLC liquid chromatography (LC) system and autosampler. The instrument is used mostly for the analysis of small molecules and metabolites.

 

MALDI-TOF/TOF Mass Spectrometer Instrument Details

The facility houses a MALDI-TOF/TOF 4800 (ABI/SCIEX) mass spectrometer used for analysis of peptides, proteins and other molecules. This instrument is able to identify proteins at sub-fmole levels with a high mass accuracy (less than 50 ppm). In order to perform the measurement, the digested protein (eigher in-gel or in-solution) will be mixed with a matri solution (CCA is most commonly used for peptide analyses) and 0.5μl of the following mixture will be spotted to a MALDI target plate. The expeiment can be performed in MS mode (TOF) with selected peptides undergoing a further MS/MS (TOF/TOF) mode to predict the sequence of the peptides. The combination of data obtained from MS and MS/MS can increase significantly the confidence of protein identification using a database search (MASCOT). The instrument can also be used for molecular weight determination of intact proteins.

The analysis of post-translational modifications (PTMs) in proteins is another important task in proteomics. MALDI-TOF/TOF is well suited to be used for the analysis of PTMs such as phosphorylation, acylation, etc. We support a license for our database search engine, MASCOT, so that we will be able to introduce any specific sort of modification upon request.

Please direct all inquiries about the use of this instrument for your research to the Center staff. Other peripheral services are also available, such as proteolytic digestion (typically trypsin), zip-tip desalting/purification, consultation on quantitation methods, 2D-gel electrophoresis and column chromatographic protein pruification/separation technologies.