In-Depth Host Cell Protein Analysis and Viral Protein Impurity Monitoring in Adeno-Associated Virus-Based Gene Therapy Products Using Optimized Wide Window Data-Dependent Acquisition Method.

Compared to other protein therapeutics, there is currently limited knowledge about the residual host cell proteins (HCPs) in adeno-associated virus (AAV)-based gene therapy products. This is primarily due to the lack of a robust and sensitive mass spectrometry-based method for HCP analysis in AAV samples. Existing liquid chromatography-mass spectrometry methods used for analyzing HCPs in therapeutic monoclonal antibodies (mAbs) often cannot be directly applied to AAVs, due to some unique characteristics of AAV samples encountered during their development such as limited sample availability/protein concentration and the presence of surfactants.

A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers.

The sensitivity of LC-MS in quantifying target proteins in plasma/tissues is significantly hindered by coeluted matrix interferences. While antibody-based immuno-enrichment effectively reduces interferences, developing and optimizing antibodies are often time-consuming and costly. Here, by leveraging the orthogonal separation capability of Field Asymmetric Ion Mobility Spectrometry (FAIMS), we developed a FAIMS/differential-compensation-voltage (FAIMS/dCV) method for antibody-free, robust, and ultrasensitive quantification of target proteins directly from plasma/tissue digests.

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.

Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues.

In-depth mapping of protein localizations in whole tissue by micro-scaffold assisted spatial proteomics (MASP).

Accurate, in-depth mapping of proteins on whole-tissue levels provides comprehensive insights into the spatially-organized regulatory processes/networks in tissues, but is challenging. Here we describe a micro-scaffold assisted spatial proteomics (MASP) strategy, based on spatially-resolved micro-compartmentalization of tissue using a 3D-printed micro-scaffold, capable of mapping thousands of proteins across a whole-tissue slice with excellent quantitative accuracy/precision. The pipeline includes robust tissue micro-compartmentalization with precisely-preserved spatial information, reproducible procurement and preparation of the micro-specimens, followed by sensitive LC-MS analysis and map generation by a MAsP app.

High-quality and robust protein quantification in large clinical/pharmaceutical cohorts with IonStar proteomics investigation.

Robust, reliable quantification of large sample cohorts is often essential for meaningful clinical or pharmaceutical proteomics investigations, but it is technically challenging. When analyzing very large numbers of samples, isotope labeling approaches may suffer from substantial batch effects, and even with label-free methods, it becomes evident that low-abundance proteins are not reliably measured owing to unsufficient reproducibility for quantification. The MS1-based quantitative proteomics pipeline IonStar was designed to address these challenges.

Parallel, High-Quality Proteomic and Targeted Metabolomic Quantification Using Laser Capture Microdissected Tissues.

Quantitative proteomics/metabolomics investigation of laser-capture-microdissection (LCM) cell populations from clinical cohorts affords precise insights into disease/therapeutic mechanisms, nonetheless high-quality quantification remains a prominent challenge. Here, we devised an LC/MS-based approach allowing parallel, robust global-proteomics and targeted-metabolomics quantification from the same LCM samples, using biopsies from prostate cancer (PCa) patients as the model system. The strategy features: (i) an optimized molecular weight cutoff (MWCO) filter-based separation of proteins and small-molecule fractions with high and consistent recoveries; (ii) microscale derivatization and charge-based enrichment for ultrasensitive quantification of key androgens (LOQ = 5 fg/1k cells) with excellent accuracy/precision; (iii) reproducible/precise proteomics quantification with low-missing-data using a detergent-cocktail-based sample preparation and an IonStar pipeline for reproducible and precise protein quantification with excellent data quality.

Current LC-MS-based strategies for characterization and quantification of antibody-drug conjugates.

The past few years have witnessed enormous progresses in the development of antibody-drug conjugates (ADCs). Consequently, comprehensive analysis of ADCs in biological systems is critical in supporting discovery, development and evaluation of these agents. Liquid chromatography-mass spectrometry (LC-MS) has emerged as a promising and versatile tool for ADC analysis across a wide range of scenarios, owing to its multiplexing ability, rapid method development, as well as the capability of analyzing a variety of targets ranging from small-molecule payloads to the intact protein with a high, molecular resolution.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths.