N端测序作为单克隆抗体常规放行分析方法的探讨

旨在探讨N端测序作为单克隆抗体常规放行分析方法的适用性。应用Edman降解法、质量肽图法对两个针对不同靶点的单抗进行N端测序,用肽图法寻找二者的特征性鉴别肽段,用离子色谱、毛细管区带电泳和成像毛细管等点聚焦电泳进行异质性分析。Edman降解法显示两个单抗轻链和重链的15个氨基酸分别完全一致,质量肽图法显示二者轻重链的T1肽段分别完全一致,而肽图法和3种异质性分析方法则可对两个抗体进行有效鉴别。由于人源化或人源单抗序列框架数量较为有限,两个单抗的N末端序列完全相同,运用Edman降解法进行N端测序是否能作为单抗的常规放行分析方法值得进一步商榷,同时上述多种方法可运用于单抗的鉴别分析,并可对其异质性进行控制,较N端测序分析更具有客观性。     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

Analysis of recombinant monoclonal antibody isoforms by electrospray ionization mass spectrometry as a strategy for streamlining characterization of recombinant monoclonal antibody charge heterogeneity

A therapeutic recombinant monoclonal antibody analyzed by cation-exchange chromatography exhibited a heterogeneous profile composed of approximately 10 isoforms. The peaks were isolated and characterized by electrospray quadrupole time-of-flight mass spectrometry (ESI-q-TOF-MS), N-terminal Edman sequencing, peptide mapping, and other techniques. Acidic (lower pI) peaks were found to represent deamidated and sialyated species. Higher pI peaks were found to contain N- and C-terminal heavy-chain variants. Biological activities of the more abundant isoforms were found to be comparable. An approach streamlining the characterization of antibody charge heterogeneity is proposed.     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

Sequencing and model structure of a Naja naja atra protein fragment.

We report the amino acid sequence of a basic protein isolated from the snake venom of Naja naja atra. An automated Edman sequencer was used to determine the 65-residue sequence, aided by electrospray ionization/mass spectrometry. Online reduction and pyridylethylation of the peptide was performed to identify the cysteine residues. Trypsin, chymotrypsin and aspartic digestions were carried out to derive peptide fragments for further sequencing. Fragmented peptides were overlapped to obtain the complete sequence. Molecular mass measurements of the whole protein and its fragments were used as a countercheck for sequence assignment. Further confirmation of the sequence was indicated by sequence homology to other snake venom neurotoxins. A molecular model of the tertiary structure was constructed based on sequence homology, and was refined by global minimization and extensive quality control algorithms. Electrostatic and hydrophobic surface calculations and molecular dynamics simulations were carried out to determine the functional properties of the molecule.     

Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma.

The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.     

Identification of the C-terminal portion of a protein by comparative peptide mapping

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.     

Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes.

The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study, we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.     

Mass balance strategy for protein sequencing. Application to a protein with an extended DNPNNP repeating motif.

The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide a challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.     

Conformational studies of a monoclonal antibody, IgG1, by chemical oxidation: structural analysis by ultrahigh-pressure LC-electrospray ionization time-of-flight MS and multivariate data analysis

We describe the development of a method in which protein oxidation by H₂O₂ followed by ultrahigh-pressure liquid chromatography (UHPLC) coupled with electrospray ionization time-of-flight mass spectrometry (ESI-ToFMS) and multivariate analysis are used to detect alterations in conformational states of proteins. In the study reported here, an IgG1 monoclonal antibody in native and denatured conformational states was oxidized by treatment with hydrogen peroxide. Peptide fragments generated by tryptic digestion were then analyzed by UHPLC-ESI-ToFMS. After reducing noise and extracting peaks from the LC–MS data using MzExplorer, software developed in-house and based on Matlab, we were able to distinguish peptides arising from the native and denatured states of the oxidized protein by principal component analysis. Peptides containing residues, which are inclined to undergo oxidation, such as methionine, are founded to be particularly important in this approach. We believe that the methodology could facilitate attempts to characterize the conformational states of recombinant monoclonal antibodies and other proteins.     

The complete amino acid sequence of a biologically active 197-residue fragment of M protein isolated from type 5 group A streptococci

The complete amino acid sequence of a peptic fragment (Pep M5) of the group A streptococcal type 5 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment, comprising nearly half of the native M molecule, is biologically active in that it has the ability to interact with opsonic antibodies as well as to evoke such an antibody response in rabbits. The sequence of Pep M5 was determined by automated Edman degradations of the uncleaved molecule and its enzymatically derived peptides. The primary peptides for Edman degradation were the arginine peptides obtained by tryptic digestion. The tryptic cleavage of Pep M5 was limited to the arginyl peptide bonds by derivatizing the epsilon-amino groups of lysine residues by reductive dihydroxypropylation. The overlapping peptides were generated by digestion of the unmodified Pep M5 with chymotrypsin, V8 protease, and subtilisin. The sequence thus established for the Pep M5 molecule consists of a total of 197 residues (Mr = 22,705). The Pep M5 protein contains some identical, or nearly so, repeating sequences: four 7-residue segments and two 10-residue segments. However, extensive sequence repeats of the kind previously reported within the partial sequence of another M protein serotype, namely Pep M24, were absent. The Pep M5 sequence is distinct from, but exhibits some homology with, the partial sequences of two other M protein serotypes, namely, Pep M6 and Pep M24. Furthermore, the 7-residue periodicity of the nonpolar and charged residues, an alpha-helical coiled-coil structural characteristic that was previously observed within the partial sequences of M proteins, was found to extend over a significant part of the Pep M5 sequence. The implication of these results to the function and immunological diversity in M proteins is discussed.     

Isolation and determination of the primary structure of a lectin protein from the serum of the American alligator (Alligator mississippiensis)

Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35 kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35 kDa protein was ~ 98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates.     

Detecting low level sequence variantsin recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5–5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274Tat 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.Key words: recombinant monoclonal antibody, cell line development, sequence variants, HPLC-UV/MS/MS, tryptic peptide mapping, Mascot error tolerant search     

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.     

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

The amino acid sequence of the ribosomal protein S8 of Escherichia coli.

The primary structure of protein S8 from the 30S subunit of Escherichia coli ribosomes has been determined by sequencing the peptides derived from tryptic, chymotryptic, thermolytic and staphylococcal protease digestion of the protein. Protein S8 has 129 amino acid residues which result in a molecular weight of 13996. The N-terminal part of the sequence up to position 68 is in complete agreement with the reported sequence data[1,2]. However, differences exist in the C-terminal half, where an additional hydrophobic tryptic peptide has been found.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Detecting low level sequence variants in recombinant monoclonal antibodies

A systematic analytical approach combining tryptic and chymotryptic peptide mapping with a Mascot Error Tolerant Search (ETS) has been developed to detect and identify low level protein sequence variants, i.e., amino acid substitutions, in recombinant monoclonal antibodies. The reversed-phase HPLC separation with ultraviolet (UV) detection and mass spectral acquisition parameters of the peptide mapping methods were optimized by using a series of model samples that contained low levels (0.5-5.0%) of recombinant humanized anti-HER2 antibody (rhumAb HER2) along with another unrelated recombinant humanized monoclonal antibody (rhumAb A). This systematic approach’s application in protein sequence variant analysis depends upon time and sensitivity constraints. An example of using this approach as a rapid screening assay is described in the first case study. For stable CHO clone selection for an early stage antibody project, comparison of peptide map UV profiles from the top four clone-derived rhumAb B samples quickly detected two sequence variants (M83R at 5% and P274T at 42% protein levels) from two clones among the four. The second case study described in this work demonstrates how this approach can be applied to late stage antibody projects. A sequence variant, L413Q, present at 0.3% relative to the expected sequence of rhumAb C was identified by a Mascot-ETS for one out of four top producers. The incorporation of this systematic sequence variant analysis into clone selection and the peptide mapping procedure described herein have practical applications for the biotechnology industry, including possible detection of polymorphisms in endogenous proteins.     

Epitopes on the major capsid protein of simian virus 40

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly beta-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.