Rigorous
validation of amino acid sequence is fundamental in the
characterization of original and biosimilar protein biopharmaceuticals.
Widely accepted workflows are based on bottom-up mass spectrometry,
and they often require multiple techniques and significant manual
work. Here, we demonstrate that optimization of a set of tandem mass
spectroscopy (MS/MS) collision energies and automated combination
of all available information in the measurements can increase the
sequence validated by one technique close to the inherent limits.
We created a software (called “Serac”) that consumes
results of the Mascot database search engine and identifies the amino
acids validated by bottom-up MS/MS experiments using the most rigorous,
industrially acceptable definition of sequence coverage (we term this
“confirmed sequence coverage”). The software can combine
spectra at the level of amino acids or fragment ions to exploit complementarity,
provides full transparency to justify validation, and reduces manual
effort. With its help, we investigated collision energy dependence
of confirmed sequence coverage of individual peptides and full proteins
on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab).
We found the energy dependence to be modest, but we demonstrated the
benefit of using spectra taken at multiple energies. We describe a
workflow based on 2–3 LC-MS/MS runs, carefully selected collision
energies, and a fragment ion level combination, which yields ∼85%
confirmed sequence coverage, 25%–30% above that from a basic
proteomics protocol. Further increase can mainly be expected from
alternative digestion enzymes or fragmentation techniques, which can
be seamlessly integrated to the processing, thereby allowing effortless
validation of full sequences.
The interactions between [(η 6 -p-cymene)Ru(H 2 O) 3 ] 2+ and Lmalic acid (H 2 mal), L-tartaric acid (H 2 tart), and their sulfurcontaining analogues DL-thiomalic acid (H 3 thiomal) and meso-2,3-dimercaptosuccinic acid (H 4 dmsa) were studied by pH potentiometry, NMR spectroscopy, and ESI-MS. The hydroxycarboxylates are potent metal ion binders and prevent hydrolysis at pH 7.4 in aqueous solution. Although H 2 mal forms mononuclear complexes with different degrees of protonation by the involvement of the alcohol/alkoxide group in addition to the carboxylate groups, the presence of [a]
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