With large proteins, the determination of the primary sequence and post-translational modifications is most efficiently done
after digestion with trypsin 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 liquid
chromatography — mass spectroscopy (LC-MS or 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. Absolute identification of the modified amino acids may require more than one enzyme digest to produce different
peptides. Some kinds of modifications that are easily identified by MS include: phosphorylation of threonine or serine; sulfation
or phosphorylation of tyrosine; deamidation of asparagine or glutamine; O- or N-linked glycosylation; oxidation of methionine
or cysteine; and N-terminal modification by formylation or prenylation. Combining enzymatic maps (tryptic mapping) with MS/MS
may identify single amino acid variants of the protein that cannot otherwise be seen.
MS is often used as part of hyphenated methods such as LC-MS, where the proteins are separated by a chromatographic method,
and the column eluant is then directed to the mass spectrometer for additional characterization. One of the common confusions
experienced when evaluating the results of MS analyses of proteins involves equating the observed size of the ion current
peak (for a particular ion species) with the amount of the species present. The size of the peak is sensitive to several things
and cannot be used for quantitation. For this reason, the use of LC separation and quantitation "front-end" to the MS allows
the relative amounts to be determined.
CONCLUSION
The application of all or some of the methods described in this article allows the characterization of an early stage protein
and determination of size, charge, purity, and primary, secondary, and tertiary structure. Other methods based on immunoreagents
can be used. However, in the earliest stages of development, antibodies may not have been raised to the protein of interest.
It is important to remember that powerful as these methods are, none of them is used to assay potency. In some cases it will
be possible to demontrate that information from one or more of these methods is directly related to potency. Nevertheless,
these methods must be accompanied by bioanalytical tools specifically designed to analyze the potency of a biologic.
Sheila G. Magil, PhD, is a consultant with BioProcess Technology Consultants, Inc, 289 Great Road, Suite 303, Acton, MA 01720, 978.266.9153
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