UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

Abstract

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody–drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.

Online Buffer Exchange Enables Automated Membrane Protein Analysis by Native Mass Spectrometry

“…Additionally, the automated nature of this technique enables the screening of other method parameters such as desolvation and MS/MS collision energies. Recent development of the UniDec Processing Pipeline or OptiMse (Thermo Scientific) supports these automated data collection approaches with high-throughput data analysis. This approach for online buffer exchange in conjunction with native MS enables higher-throughput analysis of membrane proteins in detergent and nanodiscs and can be a powerful tool for sample screening prior to structural analysis using cryo-EM.…”

Expanding Native Mass Spectrometry to the Masses

“…A third and well-established method for automated sample introduction is online buffer exchange with an LC stack as demonstrated by VanAernum et al The primary advantage of this method is that it utilizes LC instruments that are often already present in most laboratories, eliminating the need to purchase expensive dedicated auto sampling instruments such as the ones discussed in this section. This method has been deployed for nMS analysis of various samples demonstrating its robustness to handle various proteins, and protein complexes. − Additionally there is software support for this method from instrument vendors, as well as software vendors enabling users to analyze raw data in a batch-analysis mode and generate reports summarizing the results of several consecutive runs ( Protein Metrics Byos and UniDec Processing Pipeline ).…”

Gábor Transform-Based Signal Isolation, Rapid Deconvolution, and Quantitation of Intact Protein Ions with Mass Spectrometry

“…With the new automated signal selection and baseline-correction tools, the deconvolved mass spectra (“zero-charge” spectra) are easier and faster to generate and are much less sensitive to input parameters and baseline effects. We also analyze the quality of deconvolution through mass accuracy assessments, verification of charge state assignment, and comparison to other widely used deconvolution methods (an implementation of MaxEnt in the Agilent MassHunter BioConfirm v. 10.0 and the open-source, publicly available program UniDec from the Marty Group). ,,− The theory behind these deconvolution methods is described and compared elsewhere . New tools are introduced to aid in the identification of and “instant” mass determination for related peaks in charge distribution, validation of charge state assignments, and identification and removal of deconvolution artifacts.…”