Mass spectroscopy protein sequencing

At this stage each spot on the gel may contain several proteins. The spot of interest is removed by cutting the gel and dissolving with an enzyme. Then the cleaved proteins are studied by using chromatography and mass spectroscopy techniques to determine the amino acids in the proteins and their sequences. The results are compared with database and the identities of proteins are revealed. 

Although a number of assays and technologies are available to characterize and test protein molecules, such as peptide mapping, protein sequencing, carbohydrate analysis, electrophoresis, ELISA, and mass spectroscopy, they are not as definitive as the methods used for small molecule drugs. Hence, the test for similarity is not as well defined in the case of proteins. However, as... 

Mass spectroscopy (MS) methods can be used to analyze complex mixtures of proteins and peptides in minutes and with mass accuracy several orders of magnitude better than that obtained from electrophoretic methods. For proteins with a known sequence, the mass measurement accuracies are generally sufficient to identify the products resistant to proteolysis precisely and to define compact domains within proteins effectively. 

With large proteins, the determination of the primary sequence and post-translational modifications is most efficiently done after digestion with tiypsin or another protease to generate smaller peptides. In this case, the peptides are first separated by HPLC, most commonly RP-HPLC, and the column eluant is directed into the MS. In this hyphenated method, known as LC-MS or liquid chromatography-tandem mass spectroscopy (LC-MS/MS), the individual peptides are analyzed, allowing the identification of post-translational modification sites. In some cases, there are potentially multiple sites in a single peptide that may be modified. 

Plasma Proteome. The plasma proteome has been studied extensively. Plasma can be obtained readily from individuals by drawing blood using a simple procedure that is considered noninvasive. Plasma proteins have been characterized routinely by 2D gel followed by affinity chromatography, tandem mass spectroscopy, and comparison of protein sequence data in the protein databank. Some proteins such as albumin and transferrin, which are predominantly present in serum, are removed by affinity depletion chromatography to enrich the presence of other low-bundance proteins in plasma. More than 4000 proteins have been identified from human plasma, which are being developed to be used as a biomarker for several human diseases. 

The constitution of repetitive biomolecules like proteins or oligonucleotides can be detected by more sensitive methods than NMR. Thus, NMR is not the way to determine the sequence of a protein or RNA. This is simply because NMR requires some hundred nanomoles of substance of repetitive biomolecules, and other methods such as Edman degradation, mass spectroscopy, or oligonucleotide sequencing are much more sensitive. 

Various forms of tandem mass spectroscopy (MS/MS) have also been used in the analysis of biomolecules. Such instruments consist of an ionisation source (ESI or MALDI or other) attached to a first mass analyser followed by a gas-phase collision cell. This collison cell further fragments the selected ions and feeds these ions to a second mass detector. The final mass spectrum represents a ladder of fragment ions. In the case of peptides the collision cell usually cleaves the peptides at the amide bond. The ladder of resulting peptides reveals the sequence directly [496]. Thus, tandem MS instruments, such as the triple quadrupole and ion-trap instruments have been routinely applied in LC-MS/MS or ESI-MS/MS for peptide sequencing and protein identification via database searching. New configurations, which have been moving into this area include the hybrid Q-TOF [498], the MALDI-TOF-TOF [499] and the Fourier transform ion cyclotron resonance instruments [500]. 

As seen in some of the examples given above, spectroscopic methods are very much combined with other techniques to provide more information more quickly. Optical sensors using injection-molded microfluidics systems have already been on the market for 10 years (the BiaCore), but presently the combination of spectrometers or optical sensors with microfluidics, capillary electrophoresis and DNA arrays is still very much in the center of interest For instance, an optical biosensor can be combined with mass spectroscopy, allowing very rapid sequential determination of the binding constants, charge, size and sequence of a target protein [511]. 

Mass spectroscopy is increasingly being used to resolve amino acid sequences due to recent developments in this technique. There are several different methods that can be used, but basically this method determines the mass-to-charge ratio of individual amino acids of a fragmented protein. The pattern of fragmentation can be extremely complex, but peptide bonds are mostly broken to cause the fragmentation. The patterns of fragmentation are known, and this allows the sequence to be determined. 

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