Posttranslational modifications characterization using mass

The results for bacterial whole-cell analysis described here establish the utility of MALDI-FTMS for mass spectral analysis of whole-cell bacteria and (potentially) more complex single-celled organisms. The use of MALDI-measured accurate mass values combined with mass defect plots is rapid, accurate, and simpler in sample preparation then conventional liquid chromatographic methods for bacterial lipid analysis. Intact cell MALDI-FTMS bacterial lipid characterization complements the use of proteomics profiling by mass spectrometry because it relies on accurate mass measurements of chemical species that are not subject to posttranslational modification or proteolytic degradation. 

Size-based analysis of SDS-protein complexes in polyacrylamide gels (SDS-PAGE) is the most common type of slab gel electrophoresis for the characterization of polypeptides, and SDS-PAGE is one of the most commonly used methods for the determination of protein molecular masses.117 The uses for size-based techniques include purity determination, molecular size estimation, and identification of posttranslational modifications.118119 Some native protein studies also benefit from size-based separation, e.g., detection of physically interacting oligomers. 

The protein molecular mass is insufficient information for identification, but it is adequate to confirm identity therefore, MS is one of the preferred techniques for characterization and quality control of recombinant proteins and other biomolecules. In the same way, it has been used to study posttranslational modifications (like glycosylation and disulfide bonding pattern), and other processes that can modify protein mass.11... 

Top-down proteomics [1] is the identification and characterization of a mature, intact protein (or proteins) using primarily mass spectrometry (MS)-based techniques and the sequence information contained in genomic/proteomic databases. Unhke bottom-up proteomics [2-4], where a protein (or proteins) is digested into peptides prior to MS or tandem mass spectrometry (MS/MS) analysis [3,4], the top-down approach involves measuring the molecular weight (MW) of the intact, mature protein with its associated posttranslational modifications (PTMs) if any. The intact protein ion is then fragmented in the gas phase in order to determine its amino acid sequence as well as the location and identification of PTMs. From its earliest development, top-down proteomics has been primarily the domain of electrospray ionization (ESI) [5] (which generates mul-... 

The cross-reactivity between various fruits and different pollen allergens is a well-known fact and has already been studied in the 80s of the last century [19,20] using radioallergosorhent tests (RASTs). One of the first pollen allergens characterized by mass spectrometry is betv 1, the major birch pollen allergen [21]. Plasma desorption mass spectrometry (PD-MS) was used to confirm the primary structures of the intact purified protein, of all potential isoforms and some selected proteolytic peptides and to invesfigate any possible posttranslational modifications. 

In another application, UHPLC-MS technology was developed for rapid comparison of a candidate biosimilar to an innovator monoclonal antibody (mAb) (37). In this study, UHPLC-MS was developed for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for mAb products. Although the biosimilar product is expected to have the same amino acid sequence and modifications as the innovator s product, the observed intact mass by UHPLC-MS was different for the biosimilar compared to the innovator protein. Peptide mapping using UHPLC-MS/MS (38) revealed that the mass difference between the biosimilar and the innovator s product was due to a two amino acid residue variance in the heavy chain sequence of the biosimilar (Figure 8.6). 

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