PROTEIN IDENTIFICATION AT A GLANCE

Protein Isolation Kit for Urine Samples

Proteins are large biomolecules, or macromolecules, made up of polypeptides. Polypeptides are made up of amino acids. Thus, amino acids are the basic building block of proteins. There are different types of amino acids that make up proteins, and they all possess the same basic structure with differences only in their R-group. Proteins perform various functions in organisms including directing the body’s activities and acting as enzymes responsible for catalyzing metabolic reactions. They provide cell structure, regulate the body systems, replicate DNA and transport molecules and substances in and out of the cells.

One of the most common and powerful methods for detecting and identifying proteins is mass spectrometry. Mass spectrometry is an analytical tool used to determine the mass to charge ratio of molecules. It is also used for the accurate mass determination and characterization of proteins. A mass spectrometer identifies amino acids and determines the sequence in which they are arranged. Protein identification using mass spectrometry can be done in 2 different ways.

Peptide Mass Fingerprinting (PMF): Using PMF, target proteins can be isolated by SDS gel electrophoresis and digested directly with trypsin or cleaving chemicals. The proteins of interest are cleaved into smaller peptides, whose absolute masses can be accurately measured with the mass spectrometer. The masses of the resulting proteolytic or chemically cleaved peptides are then searched against a database containing known protein sequences. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, then there is likelihood that this protein is present and identified. One main advantage of this method is that it only uses masses of the peptides, so only the masses of the peptides has to be known.  A downside to this method is that it requires the database to have proteins which are already characterized.

De novo Peptide Sequencing: This method is used to determine amino acid sequence using its tandem mass spectrum. Because this method can obtain the peptide sequences without an existing database, it overcomes the shortcomings of peptide mass fingerprinting. It uses the mass difference between two fragment ions to estimate the mass of amino acid residues on a peptide backbone. It can be used to identify peptides with posttranslational modifications (PTMs), un-sequenced organisms or unknown peptide sequences, however, it cannot be used to derive complete sequence and oftentimes the sequence direction cannot be determined

Identification and quantitation of proteins are important in cancer, cardiovascular disease, and other complex disease research because it helps researchers to link specific proteins to the onset, severity, and prognosis of diseases. A disease is often caused by the absence of a functional protein, or the presence of a dysfunctional protein which results from a mutation in an individual’s DNA sequence which produces a protein having a composition slightly different from that of the normal protein it replaces.

Equipped with state-of-the-art instruments, for mass spectrometry and proteomics, the technique used in large scale protein identification and characterization, ITSI-Biosciences can provide unparalleled protein identification services in a timely, efficient and cost effective manner. Our team of Proteomics experts will provide support from experimental design to data interpretation to enable you realize the objectives of your research fast. ITSI-Biosciences offers comprehensive bio-analytical solutions, accurate results done at a fast turnaround time. Contact us today for a quote and make your protein testing and analysis stress free. You can reach us via Email; itsi@itsibio.com, Phone; 814-262-7331, or Fax; 814-262-7334.