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Protein Identification

The majority of samples submitted for protein identification/internal protein sequencing should be submitted as stained gel bands or spots. We cut out a 2DE gel spot or SDS-PAGE band and do an in-gel tryptic digestion. Upon request, a variety of other enzymes (e.g., chymotrypsin, Staphylococcal protease, etc.) may be used in place of trypsin. The gel must be stained with a mass spectrometry-compatible staining, for example colloidal Coommasie Brilliant Blue R-250 or Farmer's silver stain. The tryptic digest is then analyzed by MALDI-TOF/TOF-MS and LC-MS/MS. The sensitivity of detection is about 50 ng.

     MALDI-TOF-TOF

The analysis is typically done in positive ionization mode and is used to obtain intact protein masses for comparative tryptic peptide mapping, for screening of RP-HPLC isolated tryptic peptides prior to Edman sequencing, and for peptide biomarker discovery. Masses can be obtained on 1-500 fmol of 750-4,000 Da peptides with an average mass accuracy (external calibration) of <100 ppm (parts per million) in linear and 50 ppm in reflectron mode. A variety of biomolecules can be analyzed including oligosaccharides, gangliosides, and proteins that are up to ~250,000 Da. If the samples contain higher salt concentrations, we offer a simple desalting method when digestion products are loaded on AnchorChip targets or a C4 ZipTip desalting procedure. Peptide mass fingerprint (PMF) is compared to the masses of all theoretical tryptic peptides generated in silico by special search program. MS/MS is used to increase the likelihood of getting a database match because MS/MS of peptides gives a spectrum of fragment ions that contain information about the amino-acid sequence. Adding this information to the PMF allows Mascot to increase the statistical significance of a match. We use LOCAL Mascot for the database searching (latest database such as NCBInr, IPI). Mascot produces a statistically-based Mowse score that indicates if any matches are significant or not.

    ESI-MS/MS

This approach is amenable to analyzing oligonucleotides, peptides, proteins, lipids, and synthetic molecules containing various functional groups. The technique is sensitive in that it usually can be carried out on pmol amounts of sample. Analyses are done in either positive or negative ion mode on 100 Da to 100,000 Da compounds with a mass accuracy of ~50 ppm. Molecules such as oligos and peptides with large numbers of dibasic acids often require negative ion analysis. Samples should be free of detergents, polyethylene glycol type compounds, and salts. Samples in non-volatile buffer/salt solutions cannot be directly analyzed because the buffer/salt suppresses analyte ionization. Upon request, samples that contain salts or other non-volatile reagents may be subjected to a reverse-phase desalting on a ZipTip(R); however, this procedure may lead to significant sample loss. Salt-free samples are submitted dry or in a volatile solvent. Many different volatile solvent systems can be used (e.g., water or water/organic combinations utilizing acetonitrile, isopropanol, or chloroform/methanol). Unless requested, otherwise all samples will be introduced as a solution in 50% acetonitrile, 0.2% formic acid and will be run with positive ion detection. Since the analysis will fail if the sample is not soluble in this buffer, it is important that the solubility of the sample in 50% acetonitrile, 0.2% formic acid be confirmed prior to sample submission. Typically, 10-20 ul of a 1-10 uM protein solution is required for an analysis.

    FT-ICR MS

The ultra high resolution, high mass accuracy, interchangeable MALDI and ESI sources, and multiple fragmentation modes of this MS platform make it the instrument of choice for exact mass determinations on small molecules, comparative LC/MS proteome profiling, detection and identification of PTMs, de novo peptide sequencing, and determining accurate protein masses (with MW 5000 to ~60,000 Da). Exact mass measurement errors are within typically <2 ppm with external calibration on standards. This allows for the determination of the elemental composition of small molecule (<450 Da). Nanoelectrospray (nESI) or MALDI-FT-ICR MS is used when the sample amount is limited (<20μL at <10μM) and not overly complex (~typically 200-500 components such as peptides). For moderately complex samples (e.g., tryptic digest of 10 proteins), nano-flow HPLC can be coupled to nESI for separation prior to MS or MS/MS analysis of the eluting peptides. With its high mass accuracy and multiple fragmentation techniques [i.e., InfraRed MultiPhoton Dissociation (IRMPD), Electron Captured Dissociation (ECD), and Collision Induced Dissociation (CID)], FT-ICR-MS is well suited for identifying PTMs. Depending upon the PTM, ECD fragmentation often retains the covalently bound modification so the site of modification can be elucidated. IRMPD fragmentation is complementary to ECD in that it often (e.g., phosphorylation) shows loss of a neutral mass corresponding to the PTM.