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We were able to correct these sequences, validate the correction, and prepare reports with ease. the past to determine complete protein sequences [3,4]. Today, there are a number of algorithms and software packages available for purely mass spectrometry basedde novosequencing at the peptide level, such as PepNovo [5] pNovo [6], PEAKS [7], and Novor [8]. Typically, these algorithms predict a list of putative peptide sequences using a combination of MS/MS fragmentation data and the precursor ion mass for each peptide. Methacycline HCl (Physiomycine) A few software packages take this process one step further by combining thede novosequenced peptide information with a proteomic database search, and thus allow mapping of identified peptides onto a homologous sequence [911]. Together with manual spectra interpretation and assembly of peptides into amino acid chains,de novosequencing of proteins by mass spectrometrists is usually feasible [12], albeit resource intensive. When it comes to antibodies, the diversity in protein sequences present a particular challenge to the peptidede novosequencing and database matching strategy. Antibody sequences are fine-tuned to a particular antigen to maximize the protein-protein interactions. Given the exposure to many antigens in a lifetime, the number of unique antibody sequences in humans is estimated to be greater than ~10 billion [13]. This sequence diversity is created by the recombination events that produce the final heavy and light chain sequences of an antibody, as well as somatic hypermutations during affinity maturation, and isotype switching to modulate the effector function. As a result, each immune reaction produces unique sequences, which are fathomably not available in proteomic databases. The strategy employed today tode novosequence antibodies involves interpretation of spectra by experts, iterative searches facilitated by humans, and manual sequence assembly. An alternative strategy is usually using orthogonal methods, such as phage display, in conjunction with mass spectrometry to sequence antibodies [14]. However, these actions incur a heavy cost on the time needed to complete an analysis, which in turn prevents antibodyde novosequencing from being applicable to high-throughput applications. Here, we describe the methodology and a new algorithm, Supernovo(Protein Metrics Inc.), for automatedde novosequencing of monoclonal antibodies. We applied a combination of peptidede novosequencing, proteomic database searching with wildcards,in silicorecombination of antibody coding exons, and final sequence assembly from identified peptides to automatically sequence monoclonal antibodies using bottom-up proteomics. We discuss the novel and high-throughput applications in the biopharmaceutical industry, which are enabled by the hands-free nature of the sequence determination process. == Methods == == Sample Preparation == Monoclonal antibody samples were diluted to 1 1 mg/mL concentration using ultrapure phosphate buffered saline answer pH 7.4 (VWR) and denatured by adding 360ul of 8M Gunadine HCl, 4mM EDTA answer (Sigma-Aldrich). 25mM DTT was added and the sample was incubated at 37C Rabbit Polyclonal to CLK4 for 1 hour to reduce disulfide bonds. Reduced antibody was then alkylated with 60 mM iodoacetamide or iodoaceticacid (Sigma-Aldrich) at room temperature Methacycline HCl (Physiomycine) for 30 minutes. At the end of alkylation, 37 mM DTT was added to quench unreacted iodoacetamide. Sample was Methacycline HCl (Physiomycine) immediately buffer exchanged into 50 mM Tris, 1mM CaCl2, pH 8.0 using Zeba spin desalting columns (Thermo Scientific). Proteolytic digestion was carried out by addition of enzyme (sequencing grade Trypsin, Promega/Chymotrypsin, Roche/Lys-C, Promega/Pepsin, Promega) at a 1:20 enzyme to protein ratio. The trypsin and Chymotrypsin digest samples were incubated at room temperature and the Lys-C digest sample was incubated at 37C over night Methacycline HCl (Physiomycine) with gentle agitation. All the samples were acidified to 1% final TFA after enzymatic digestion. For the pepsin digest, sample was acidified to pH 2 and the digestion was allowed to proceed at 37C for 1 hour. Pepsin digest was inactivated by heating the sample at 95C for 15 minutes. The antibody digests were desalted as described [15]. Briefly, 30l slurry of R2 20m Poros beads (Life TechnologiesCorporation) was added to each sample and the Methacycline HCl (Physiomycine) samples were incubated at 4C with agitation for 2 hours. The beads were loaded onto the vacant TopTip (Glygen) using a microcentrifuge for 30 sec at 1500 RPM. The sample vials were rinsed three times with 0.1% TFA and each rinse was added to the corresponding Toptip followed by microcentrifugation. Extracted poros beads were further rinsed with 0.5% acetic acid. Peptides were eluted by addition of 40% acetonitrile in 0.5% acetic acid followed by the addition of 80% acetonitrile in 0.5% acetic acid. The organic solvent was removed using a SpeedVac concentrator and the samples were reconstituted in 0.5% acetic acid. == Liquid Chromatography/Mass Spectrometry == Agilent AdvanceBio peptide mapping column.