Sample preparations protein identification

Here, we will describe a range of applications of MALDI-MS, from the concepts of in-depth analysis of purified proteins to applications of MALDI-MS in a broader, proteomics-based research where proteins are identified, characterized, and quantified. In addihon, issues of sample preparation, protein characterization and identification strategies and bioinformatic tools for data interpretation wiU be discussed. The concepts of peptide fragmentation, sequencing and derivatization, analysis of post-translational modifications and the clinical apphcations of MALDI-MS are also briefly outlined. 

As noted above, whole-cell MALDI-TOF MS was intended for rapid taxonomic identification of bacteria. Neither the analysis of specific targeted bacterial proteins, nor the discovery of new proteins, was envisioned as a routine application for which whole cells would be used. An unknown or target protein might not have the abundance or proton affinity to facilitate its detection from such a complex mixture containing literally thousands of other proteins. Thus, for many applications, the analysis of proteins from chromatographically separated fractions remains a more productive approach. From a historical perspective, whole-cell MALDI is a logical extension of MALDI analysis of isolated cellular proteins. After all, purified proteins can be obtained from bacteria after different levels of purification. Differences in method often reflect how much purification is done prior to analysis. With whole-cell MALDI the answer is literally none. Some methods attempt to combine the benefits of the rapid whole cell approach with a minimal level of sample preparation, often based on the analysis of crude fractions rather... 

The ELISA can be used for identification and quantitation of the protein product (biopharmaceutical) of interest throughout the development, production, and manufacturing process. For example, in the initial development phase, ELISAs can aid in the selection of the best cell line. In the early manufacturing steps, it can be used to identify the appropriate product-containing pools or fractions in process to be subjected to further purification. Because of the selectivity of ELISA, it is a suitable tool to select out the protein of interest from complex protein mixtures, such as cell culture fermentation media or product pools in early steps of protein recovery as well as downstream processing. Even complex mixtures do not require much sample preparation. It is important to determine... 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

Traditional biochemical techniques such as liquid chromatography (LC), gel electrophoresis, capillary electrophoresis (CE), and mass spectrometry (MS) have been widely used for the complete analysis of salivary proteins and peptides. The recent advances in these technical approaches applied to peptidomics have allowed a better comprehensive analysis of peptides in human whole saliva, envisioning the identification of potential salivary biomarkers of oral and systemic diseases. Sample preparation is a critical experimental step for the successful identification of peptides using MS-based approaches, for their quantitation and identification of PTMs. 

Crews et al. [81] studied cooked cod by means of a two-step in vitro gastrointestinal enzymolysis. For the first step of sample preparation they employed gastric juice (1 percent m/v pepsin, pH = 2.0, in 0.15 mol l-1 NaCl) at 37°C for 4 h. Afterwards a pancreatin-based mixture was added to the sample solution containing 1.5 percent m/v pancreatin, 0.5 percent m/v amylase, and 0.15 percent m/v bile salts in 0.15 mol l-1 NaCl at pH = 6.9 for a further 4 h at 37°C. The relatively short (8 h) enzymatic activity and the lack of enzymes capable of hydrolyzing proteins directly into amino acids resulted in the identification of inorganic Se (IV) only, as no selenoamino acids could be detected. 

Scheler, C., Wittmann-Liebold, B., Jungblut, P. (1996). Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry. Electrophoresis 17,... 

The same concept in proteomics studies has technological implications, e.g., which method, sample preparation protocols, and instrumentation will be used. Again, top-down analysis will be based on isolation, analysis, and characterization of an intact protein to reveal its function. Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR) (Marshall et al., 1998) facilitates such approach in protein identification as a result of random fragmentation of an intact molecule. In contrary, bottom-up approach is based on up-front fragmentation of the protein in question using various proteolytic enzymes with known specificity (Chalmers et al., 2005 Millea et al., 2006). In these experiments, trypsin is most commonly used. An important question that remains is whether more... 

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