RESUMEN
At present, mass spectrometry provides a rapid and sensitive means for making conclusive protein identifications from complex mixtures. Sequencing tryptic peptides derived from proteolyzed protein samples, also known as the "Bottom Up" approach, is the mass spectrometric gold standard for identifying unknowns. An alternative technology, "Top Down" characterization, is emerging as a viable option for protein identifications, which involves analyzing the intact unknowns for accurate mass and amino acid sequence tags. In this paper, both characterization methods were employed to more comprehensively differentiate two early-eluting peaks in a process-scale size-exclusion chromatography (SEC) step for a recombinant, immunoglobulin gamma-1 (IgG-1) fusion protein. The contents of each SEC peak were enzymatically digested, and the resulting peptides were mapped using reversed-phase (RP) HPLC-ion trap MS. Many low-level UV signals were observed among the fusion protein-related peptide peaks. These unknowns were collected, concentrated, and analyzed using nanoelectrospray (nanoES) collision-induced dissociation (CID) tandem (MS/MS) mass spectrometry for identification. The peptide sequencing experiments resulted in the identification of twenty host cell-related proteins. Following peptide mapping, the contents of the two SEC peaks were protein mass profiled using on-line RP HPLC coupled to a high-resolution, quadrupole time-of-flight (Qq/TOF) MS. Unknown proteins were also collected, concentrated, and dissociated using nanoES CID MS/MS. Intact protein CID experiments and accurate molecular weight information allowed for the identification of three full length host cell-derived proteins and numerous clips from these and additional proteins. The accurate molecular weight values allowed for the assignment of N- and C-terminal processing, which is difficult to conclusively access from peptide mapping data. The peptide-mapping experiments proved to be far more effective for making protein identifications from complex mixtures, whereas the protein mass profiling was useful for assessing modifications and distinguishing protein clips from full length species.
Asunto(s)
Fragmentos de Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Análisis de Secuencia/métodos , Alquilación , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina G/química , Ratones , Mapeo Peptídico , Receptores de Interleucina/química , Proteínas Recombinantes de Fusión/análisisRESUMEN
An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein-ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.
Asunto(s)
Técnicas de Química Analítica , Proteínas/química , Amidas/química , Frío , Bases de Datos de Proteínas , Medición de Intercambio de Deuterio , Hormona de Crecimiento Humana/química , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Selective inhibition of cyclooxygenase-2 (COX-2) leads to relief of pain and inflammation with reduced gastrointestinal side effects relative to nonsteroidal anti-inflammatory drugs. 2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a selective COX-2 inhibitor that covalently modifies the protein by acetylating Ser-530. We utilized site-directed mutants in the COX-2 active site to probe the molecular determinants of APHS acetylation of COX-2. Incorporation of acetyl groups into Ser-530 was monitored by HPLC and mass spectrometry. Mutations that introduce steric bulk into a channel at the top of the active site (e.g., G533A, G533V) lead to a significant reduction in APHS acetylation. Reduction in acetylation is also observed by mutation of the active-site tyrosine (Tyr-385) to phenylalanine. Mutations in the side-pocket region, into which diarylheterocycle inhibitors insert, do not affect the ability of APHS to acetylate COX-2. Surprisingly, mutation of Arg-120, which is located on the floor of the active site, strongly reduces acetylation. Based on these results, we propose that the heptynyl side chain of APHS inserts into the top channel and acetylates Ser-530 with the assistance of hydrogen bonding from Tyr-385. Arg-120 is proposed to fix the conformation of the active site to one that favors acetylation.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Heptanos/farmacología , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Sulfuros/farmacología , Alquinos , Secuencia de Aminoácidos , Animales , Arginina/química , Sitios de Unión , Línea Celular , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno/farmacología , Ciclooxigenasa 2 , Hidrógeno/metabolismo , Enlace de Hidrógeno , Insectos , Ligandos , Espectrometría de Masas , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tirosina/química , Tirosina/metabolismoRESUMEN
Identifying unknown proteins has become a central focal point for proteomic and biopharmaceutical development laboratories. Our laboratory investigated using quadrupole time-of-flight mass spectrometry (Qq/TOFMS) for the analysis of intact proteins for the purpose of identifying unknowns while limiting the number of sample-handling steps between protein extraction and identification. Eight standard proteins, both unmodified and disulfide-bonded and ranging in mass from 5 to 66 kDa, were analyzed using nanoelectrospray and collision-induced dissociation to generate peptide sequence tags. An MS analysis, followed by MS/MS analyses on two to five individual protein charge states, were obtained to make an identification. Peptide sequence tags were extracted from the MS/MS data and used, in conjunction with molecular mass and source origin, to obtain protein identifications using the web-based search engine ProteinInfo (www.proteometrics.com). All of the proteins were unambiguously identified from the input data, after which, all of the major product ions were identified for structural information. In most cases, N- and/or C-terminal ions, and also stretches of consecutive product ions from the protein interior, were observed. This method was applied to the analysis and identification of an unknown detected via reversed-phase high-performance liquid chromatography.