High-Throughput Protein Sequencing

Abstract

We have designed and implemented an autosampler that provides additional sample capacity on a commercial protein sequencer. The autosampler attaches to a standard ABI Procise sequencer, enabling a single-sample cartridge to hold up to six separate samples. The autosampler is used in combination with faster Edman cycles and a rapid 12-min PTH separation to significantly increase the speed of automated protein sequencing. We also describe Edman chemistry modifications that improve coupling efficiency and prevent internal cleavage that can occur when samples are sequenced in the standard glass cartridges.

Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery

“…Edman degradation[1] and a combination of Edman degradation with mass spectrometry [2] have been used successfully in the past to determine complete protein sequences [3, 4]. Today, there are a number of algorithms and software packages available for purely mass spectrometry based de novo sequencing at the peptide level, such as PepNovo [5] pNovo [6], PEAKS [7], and Novor [8].…”

A Platform for Characterizing Therapeutic Monoclonal Antibody Breakdown Products by 2D Chromatography and Top-Down Mass Spectrometry

“…Additionally, subtle modifications resulting from degradation (e.g., methionine oxidation) required relatively low throughput peptide mapping experiments to monitor individual sequences affected. Despite advancements in high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography (22) and sequencing technologies (23), routine analysis requires significant time resources.…”

“…Using high-resolution collisionally induced and electron transfer dissociation methods, additional identifications were made with specific localization of unpredicted modifications. As examples, a modified Fab fragment (N-and C-terminal cyclization, 47,902 Da) and a hydrolyzed free light chain impurity components (23,191 Da) were identified with a high degree of confidence (E value, <1e-5). This work describes the approach for top-down characterization of breakdown products and is readily applicable to additional monoclonal antibodies (mAb) characterization experiments, including charge isoform characterization and aggregate analysis, for a more thorough understanding of therapeutic mAb drug products.…”

Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins

“…Many analytical advancements have emerged to satisfy researchers' needs, including the development of rapid chromatography techniques [13], higher resolution mass spectrometers [14], more sensitive and high-throughput N-terminal sequencers [15,16], and faster search engines for sequence algorithm processing and data analysis [17]. As well as these breakthroughs in instrumentation and informatic-based technologies, more traditional bench-top biochemical techniques are also continuously developing, for example, the area of proteolytic digestion.…”