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Bottom-Up Measurements

In bottom-up methods, the protein is digested into small peptides by a protease (e.g. trypsin, chymotrypsin, AspN, GluC, or LysC), followed by LC-MS or LC-MS/MS analysis (Figure 1).  Proteolytic digestion, combined with LC-MS/MS analysis is the most sample-consuming, time-consuming, and labor intensive of all the MS analysis types.  Proteolytic digestion can result in loss of labile modifications, such as succinimide, and amino acid rearrangements, which could lead to incorrect or incomplete structural characterization.4

Despite the complications, bottom-up methods have been used successfully to confirm protein sequences, characterize post-translational modifications and other modifications that may be related to the manufacturing process or storage, as well as for the characterization of disulfide linkages.

 

Figure 1: Bottom-up analysis.

mAb proteolytic digestion can be performed with trypsin, after reduction and alkylation, under denaturing conditions.  Alternatively, non-reduced trypsin or LysC proteolysis can also be used, followed by disulfide reduction.  This may be preferred due to its simplicity and ability to generate both reduced and non-reduced peptides in a single experiment.  Trypsin is the most commonly used enzyme for proteolytic digestion, however, secreted aspartic protease 9 (Sar 9) which generates larger fragments (3.5 kDa average) is a promising alternative as it introduces less sample preparation artifacts (e.g. deamidation) due to the lower digestion pH.5  Other less often used proteases include Glu-C (V8) and Asp-N.  Chemical cleavage can be carried out using cyanogen bromide (CNBr).

All of these methods produce peptides that fragment efficiently in MS/MS experiments.  Large peptides (>4 kDa) are difficult to characterize by MS/MS and very small peptides (2-3 residues) are often lost due to lack of retention on reversed phase HPLC columns (this is a situation that can be remedied by the use of hydrophilic interaction liquid chromatography, HILIC).

Bottom-up methods utilize reversed phase (RP) LC or capillary zone electrophoresis (CZE) coupled to MS.  These techniques do not resolve all peptides in a complex protein digest, therefore, additional separation, based on m/z, in high-resolution Q-ToF or Orbitrap instruments significantly increases sequence coverage and allows low level PTMs to be highlighted.

Data acquired during MS/MS analyses are used to sequence the protein which provides further confidence in identification of PTM types and locations.  CID is commonly used as the mode of fragmentation as it produces predictable fragments along the peptide bond to produce y- and b-ions which contain the peptide C- and N-terminus respectively (Figure 2).  The mass difference between successive y- and b-ions corresponds to the residual mass of the amino acids.  The predictable spectra that arise from these experiments allow automated data analysis, with most instrument manufacturers providing powerful biopharmaceutical supporting software as part of their portfolio.

Figure 2: b and y fragment ions.

 

CID maintains stable modifications, such as oxidation and deamidation, on the peptide backbone.  However, labile modifications, such as glycosylation and phosphorylation, are preferentially cleaved making identification of the modification site difficult.  In this case, ECD or ETD should be selected instead, as these modes of fragmentation preserve these labile modifications.  Furthermore, ECD and ETD generate c- and z-ions via cleavage of the N-Cα backbone bonds; these different ion types can provide complementary information for structural characterization (Figure 3).

Figure 3: c and z fragment ions.

 

Bottom-up measurements can be used to characterize post-translational, in-process, and in-storage modifications, and degradations, including:

  • Heavy chain C-terminal processing
  • N-terminal cyclization
  • Asparagine deamidation, succinimide formation, and aspartic acid isomerization
  • Fragmentation
  • Oxidation
  • Non-enzymatic glycation
  • Alternative signal-peptide cleavage sites
  • O-linked glycosylation
  • Cysteinylation
  • Disulfide exchange
  • Non-reduced thioether cross-linking or heavy and light chains
 

 

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