RESUMEN
Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.
Asunto(s)
Aminoácidos , Péptidos , Aminoácidos/química , Aminoácidos/análisis , Péptidos/química , Péptidos/análisis , Espectrometría de Masas/métodos , Cromatografía por Intercambio Iónico/métodos , Hidrólisis , Ninhidrina/químicaRESUMEN
The aim of this chapter is to give a short introduction to peptide analysis by mass spectrometry (MS) and interpretation of fragment mass spectra. Through examples and guidelines, we will demonstrate how to understand and validate search results and how to perform de novo sequencing based on the often very complex fragmentation pattern obtained by tandem mass spectrometry (also referred to as MSMS). The focus will be on simple rules for interpretation of MSMS spectra of tryptic as well as non-tryptic peptides.
Asunto(s)
Péptidos , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Péptidos/análisis , Péptidos/química , Proteómica/métodos , HumanosRESUMEN
In the quality control of synthetic peptides, mass spectroscopy (MS) serves as an optimal method for evaluating authenticity and integrity. Typically, the sequence of a synthetic peptide is already established, thereby directing the focus of analysis towards validating its identity and purity. This chapter outlines straightforward methodologies for conducting MS analyses specifically tailored for synthetic peptides.
Asunto(s)
Espectrometría de Masas , Péptidos , Péptidos/química , Péptidos/análisis , Espectrometría de Masas/métodos , Control de Calidad , Secuencia de Aminoácidos , Espectrometría de Masas en Tándem/métodosRESUMEN
Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogendeuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.
Asunto(s)
Mapeo Epitopo , Radical Hidroxilo , Huella de Proteína , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Mapeo Epitopo/métodos , Huella de Proteína/métodos , SARS-CoV-2/inmunología , SARS-CoV-2/química , Radical Hidroxilo/química , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Unión Proteica , COVID-19/virología , COVID-19/inmunología , Sitios de Unión , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/química , Espectrometría de Masas/métodos , Dominios ProteicosRESUMEN
Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.