Sequencing, proteins peptide mapping

Disulphide bonds (or bridges), whether already present in the native protein or artificially introduced, must be cleaved before carrying out amino acid analysis or sequencing or peptide mapping (Creighton 1989). This can be done in various ways. Oxidative cleavage with performic acid ... 

In practice, it is rarely possible to sequence lengths of more than 20 or so amino acids by these methods because the mixtures become too complex, and degradation patterns are uneven. However, a range of short sequences from a collection of different peptides from the one protein ( peptide mapping ) is often sufficient to identify the protein. If the different peptide sequences overlap, then the entire protein sequence can be determined. 

Proteins are amino acid chains that fold into unique three-dimensional structures. The primary structure of proteins is the peptide sequence of the whole molecule, which can be characterized by the amino acid composition, N-terminal amino acid sequence, C-terminal amino acid sequence, and peptide mapping. 

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). 

Protein sequencing by creation of overlap peptides (mapping),... 

Peptide mapping The process of considering the amino acid sequence information from peptides obtained by enzyme digestion in an attempt to derive the (amino acid) sequence of the parent protein. 

The G-protein that has been termed Gp, and that is linked to phospholipase C activation, may in fact be Gaj 2 or Gc. 3. Ga is designated as the G-protein responsible for activation of phospholipase A2, which results in arachidonic acid release. Some experimental evidence indicates that, at least in HL-60 cells, different agonists can preferentially activate different phospholipases, and some of these are responsible for the activation of secretion. In neutrophils, the two pertussis-toxin-sensitive Ga-proteins (Gaj-2 and G j 3) have been identified by peptide mapping of proteolytic digests of the proteins, by peptide sequencing and by immunoblotting. Complementary-DNA clones for the mRNA of these two molecules have also been isolated from an HL-60 cDNA library. Gai-2 is five to ten times more abundant than Gai.3, the former component comprising 3% of the total plasma membrane proteins. It is possible that these two different Ga-subunits are coupled to different phospholipases (e.g. phospholipases C and D). Pertussis toxin inhibits the secretion of O2 after stimulation of neutrophils by fMet-Leu-Phe, but pertussis-toxin-insensitive G-proteins are also present in neutrophils. These may be members of the Gq family and may be involved in the activation of phospholipase Cp (see 6.3.1). 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

The primary analytical applications of RPLC in the development of biopharmaceuticals are the determination of protein purity and protein identity. Purity is established by analysis of the intact protein, and RPLC is useful in detecting the presence of protein variants, degradation products, and contaminants. Protein identity is most often established by cleavage of the protein with a site-specific protease followed by resolution of the cleavage products by RPLC. This technique, termed peptide mapping, should yield a unique pattern of product peptides for a protein that is homogeneous with respect to primary sequence. 

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

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... 

The distinguishing feature of CS H2A is a nine amino acid extension on its C-terminus. The possibility that CS H2A is the sea urchin H2A.X has been raised on the basis of its structure and its similarity to a large H2A stored in Xenopus eggs that was identified as H2A.X [127] this Xenopus protein has been identified as H2.X on the basis of its position in two dimensional gels and its peptide map [129], but its sequence has not been determined. The C-terminal sequence of CS H2A (SMEY) resembles the SQ(ED)(ILFY) consensus of H2AX. The Xenopus and sea urchin proteins also show similar phosphorylation patterns during chromatin assembly [128,129]. Additional studies will be required to determine the relationship, if any, between CS H2A and H2A.X. 

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