RESUMO
A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3' specificities of PPEP-1 and PPEP-2, while identifying the endogenous substrate of PPEP-2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.
RESUMO
Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-ßâ¯-â¯1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2⯵L. HILIC-MS of rhIFN-ßâ¯-â¯1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72.
Assuntos
Eritropoetina/análise , Interferon beta-1a/análise , Cromatografia Líquida de Alta Pressão , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Polissacarídeos/análise , Proteínas Recombinantes/análiseRESUMO
A high-throughput sample preparation protocol based on the use of 96-well molecular weight cutoff (MWCO) filter plates was developed for shotgun proteomics of cell lysates. All sample preparation steps, including cell lysis, buffer exchange, protein denaturation, reduction, alkylation and proteolytic digestion are performed in a 96-well plate format, making the platform extremely well suited for processing large numbers of samples and directly compatible with functional assays for cellular proteomics. In addition, the usage of a single plate for all sample preparation steps following cell lysis reduces potential samples losses and allows for automation. The MWCO filter also enables sample concentration, thereby increasing the overall sensitivity, and implementation of washing steps involving organic solvents, for example, to remove cell membranes constituents. The optimized protocol allowed for higher throughput with improved sensitivity in terms of the number of identified cellular proteins when compared to an established protocol employing gel-filtration columns.