Protein identification is most commonly accomplished by proteolytic digestion followed by LC-MS/MS analysis. When applicable, N-terminal Edman sequencing is also available. After an in-depth project discussion, the sample is prepared by the user following our advised protocols, and submitted to the facility for analysis. Samples are enzymatically digested, run on nano-capillary HPLC/MSMS, and the MSMS spectra are correlated against a specific database for peptide identification.
Complex Mixture Analysis
Complex mixtures of proteins are identified by a number of single- and multi-dimensional approaches. For example, GeLC, in which an entire lane of an SDS-PAGE gel is excised into sections, affords the user a two dimensional separation of the protein mixture based on protein intact molecular weight (SDS-PAGE) and then individual peptide hydrophobicity by reversed phase chromatography (RPLC). A similar method known as MUDPit (Multidimensional Protein Identification Technology) starts with a solution digestion of the sample, then two dimensional chromatography by strong cation exchange chromatography (SCX) followed by reversed phase chromatography (RPLC).
Posttranslational Modification Site Determination
Starting with a single highly purified protein in an SDS-PAGE gel slice, multiple sites of modification, eg. phosphorylation, acetylation and others, can be determined. This process involves a detailed project discussion and careful selection of multiple enzymes to maximize peptide coverage for specific sites of interest.
One of the major challenges in modern proteomics is characterizing the differences in protein expression between two or more samples in a statistically relevant method. For instance, these methods could show differences in protein expression between treated and non-treated cell lines, healthy and sick animals, or between knockout and wild type organisms.
Labeled: Quantitative mass spectrometry normally utilizes stable isotope labeling at the whole cell level, intact protein level or even peptide level. There are several well established techniques to do this, and a detailed project consultation prior to beginning an experiment with this goal is mandatory.
- SILAC (stable isotope labeling with amino acids in cell culture) is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometry (MS)-based quantitative proteomics. SILAC relies on metabolic incorporation of a given ‘‘light’’ or ‘‘heavy’’ form of the amino acid into the proteins. The method relies on the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, 13C, 15N). Thus in an experiment, two cell populations are grown in culture media that are identical except that one of them contains a ‘light’ and the other a ‘heavy’ form of a particular amino acid (e.g. 12C and 13C labeled L-lysine, respectively). When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After a number of cell divisions, each instance of this particular amino acid will be replaced by its isotope labeled analog. Since there is hardly any chemical difference between the labeled amino acid and the natural amino acid isotopes, the cells behave exactly like the control cell population grown in the presence of normal amino acid. It is efficient and reproducible as the incorporation of the isotope label is 100%, however is generally applicable only to cell or tissue culture experiments due in large part to the expense of stable isotope labeled growth media.
- TMT (tandem mass tags) Isobaric chemical tags are powerful tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. They are small chemical molecules with identical structure that covalently attach to the free amino termini of lysine residues of peptides and proteins, thereby labeling various peptides in a given sample. During the MS/MS analysis, each isobaric tag produces a unique reporter ion signature that makes quantitation possible. In a typical MS analysis, the labeled peptides are indistinguishable from each other; however, in the tandem MS mode during which peptides are isolated and fragmented, each tag generates a unique reporter ion. Protein quantitation is then accomplished by comparing the intensities of the six reporter ions in the MS/MS spectra.
- Di-Methyl labelling analysis - uses stable isotope incorporation at the peptide level using fairly inexpensive reagents and is applicable to most samples.
- ICAT (Isotope Coded Affinity Tags) is a well-publicized method of relative quantification in which two or more different samples are labeled at the intact protein level with isotope coded tags. The samples are combined for digestion, and the labeled peptides are specifically removed by affinity chromatography to one part of the label (biotin-avidin enrichment). These labeled peptides can then be run together, with isotopically different peptides eluting near each other at a mass difference of a couple of amu in the full MS scan for quantitative comparison of expression levels between samples. A newer version has acid labile labels, which has improved the chromatographic performance of the process. This process has the advantage of labeling intact proteins prior to digestion, which can reduce system variability.
- iTRAQ (Isotope Tags for Relative and Absolute Quantitation) is another popular technique that includes up to 10 isotopic labels for multiplexing experimental variables. The technique is based upon chemically tagging the N-terminus of peptides generated from protein. The labeled samples are then combined (post labeling), fractionated by nano-LC and analyzed by tandem mass spectrometry. Peptides are chromatographically resolved as single peaks with identical full MS masses. Fragmentation of the labeled peptides generates a low molecular mass reporter ion that is unique to the tag used to label each of the samples. Measurement of the intensity of these reporter ions, enables relative quantification of the peptides in each digest and hence the proteins from where they originate. This process has the advantage of no chromatographic interference from the labels but requires a low mass MSMS scan to observe the reporter ions.
- AQUA – method of absolute quantitation based on synthetic "heavy" peptides that is used as an absolute standard. A fundamental goal of cell biology is to define the absolute levels of every protein expressed by an organism under the conditions of interest. Precise measurement of protein changes in terms of molecules per cell, and for all expressed components, would provide the high quality datasets necessary for a comprehensive understanding of disease at the molecular level. Absolute quantification of proteins uses 13C- and 15N- labeled synthetic reference peptides and tandem MS to measure expression in terms of number of molecules per cell. This process targets specific peptides individually and can become expensive but provides the most exact quantitation in many cases. Typically a SIM or SRM experiment is preformed on the mass spectrometer to target only the peptides of interest, so the method can be adapted to high throughput proteomics experiments.
Label-free methods for quantitation have recently become popular and shown good results in blind studies that have been published. These processes rely on highly reproducible chromatography; typically with high pressure sub-2 micron particle reverse phase columns and traps, to produce statistically relevant data. The Q-TOF premier is one system that targets this type of analysis directly, with the Protein Expression System and nano-Acquity UPLC. This system eliminates the isolation step of MSMS data acquisition, relying on post-run analysis to construct individual MSMS spectra from the mix of MSMS data.
N-terminal Edman Sequence Analysis
N-terminal sequence analysis is a chemical method in which the amino terminal amino acid is labeled with phenylisothiocyanate and specifically cleaved, followed by identification of the released phenylthiohydantoin amino acid by RPLC. This process can sequence upwards of 30 amino acids given sufficient quantities, typically 10pmol or more, of a single protein. Edman sequencing affords the researcher the ability to characterize the N-terminus of a protein directly, including quantitatively differentiating between even single amino acid cleavage sites. This process gives true de-novo sequence information, as it is not a database dependent technique, and has been an established method in protein research for many decades.
C-terminal Sequence Analysis
In this lab, we use multiple enzymes to obtain redundant peptides which exhaustively define the C-terminal region of a purified protein. Multiple instrument runs are combined with custom bioinformatics tools to provide the final result.
De novo Sequence Analysis
All of the previously mentioned mass spectrometry techniques rely on the protein sequence being known and available for comparison of mass spectra to a database. If this is not the case, identical peptides from homologous proteins can often be found, leaving many still unidentified. While software algorithms have advanced, these spectra often require expert manual interpretation. This laboratory has over 40 years of combined experience specializing in de novo interpretation.
Intact Protein Molecular Weight Determination by ESI/LC/TOF
Molecular weight determination of intact protein can be done by direct infusion Electro Spray Ionization (ESI) or Liquid Chromatographic (LC) on the Agilent 6210 ESI-TOF. A concentrated solution of desalted protein is sprayed into the instrument, with a series multiple charge state envelopes the expected result. This data is then deconvoluted to intact protein mass measurement, typically up to 200kDa, depending on the homogeneity of the sample.
Analysis of mammalian plasmas with depletion of IgG and Albumin protocol
Quantitative analysis of plasma proteomes after a depletion for IgG and Albumin protocol
Please call to discuss any of the above and we will be happy to work with you on these projects