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Jason C Rouse

Publications and source records attributed to Jason C Rouse.

5 recordsLinked to original sources
PubMed2006

Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain.

A unique, late-eluting "basic peak" (relative to the "main peak") was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a top-down characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversed-phase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or alpha-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and alpha-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal alpha-amidation in mAbs has not been reported previously.

Amides↗
PubMed2002

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody.

Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase-high-performance liquid chromatography mass spectrometry (RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.

Amino Acid Sequence↗
PubMed2002

Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry.

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.

Amino Acid Sequence↗
PubMedDate not supplied

"Top Down" characterization is a complementary technique to peptide sequencing for identifying protein species in complex mixtures.

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.

Alkylation↗