Biology subjects

Huguet, R.

Publications and source records attributed to Huguet, R..

6 recordsLinked to original sources

bioRxiv2023

Exposing the molecular heterogeneity of glycosylated biotherapeutics

Glycosylated biotherapeutics are an emerging class of drugs with high molecular heterogeneity, which can affect their safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins. Here, we present a new approach to glycoform fingerprinting that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact glycoproteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment and offers insights into glycoform composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drugs pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

biochemistry↗

bioRxiv2022

Real-time library search increases cross-link identification depth across all levels of sample complexity

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

systems biology↗

bioRxiv2021

A novel multifunctional role for Hsp70 in binding post-translational modifications on clients

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically-important PTMs on client proteins. O_LIIn vivo confirmation of Hsp70 dimerization C_LIO_LIComprehensive direct interactome of Hsp70 C_LIO_LIMulti-domain interactions between Hsp70 and client proteins C_LIO_LIIdentification of novel biologically-important client protein PTMs C_LI

molecular biology↗

bioRxiv2021

Real-Time Search Assisted Acquisition on a Tribrid Mass Spectrometer Improves Coverage in Multiplexed Single-Cell Proteomics

In the young field of single-cell proteomics (scMS), there is a great need for improved global proteome characterization, both in terms of proteins quantified per cell and quantitative performance thereof. The recently introduced real-time search (RTS) on the Orbitrap Eclipse Tribrid mass spectrometer in combination with SPS-MS3 acquisition has been shown to be beneficial for the measurement of samples that are multiplexed using isobaric tags. Multiplexed single-cell proteomics requires high ion injection times and high-resolution spectra to quantify the single-cell signal, however the carrier channel facilitates peptide identification and thus offers the opportunity for fast on-the-fly precursor filtering before committing to the time intensive quantification scan. Here, we compared classical MS2 acquisition against RTS-SPS-MS3, both using the Orbitrap Eclipse Tribrid MS with the FAIMS Pro ion mobility interface and present a new acquisition strategy termed RETICLE (RTS Enhanced Quant of Single Cell Spectra) that makes use of fast real-time searched linear ion trap scans to preselect MS1 peptide precursors for quantitative MS2 Orbitrap acquisition. We show that classical MS2 acquisition is outperformed by both RTS-SPS-MS3 through increased quantitative accuracy at similar proteome coverage, and RETICLE through higher proteome coverage, with the latter enabling the quantification of over 1000 proteins per cell at a MS2 injection time of 750ms using a 2h gradient.

biochemistry↗

bioRxiv2020

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

systems biology↗

bioRxiv2019

Direct Measurement of Light and Heavy Antibody Chains Using Differential Ion Mobility Spectrometry and Middle-Down Mass Spectrometry

The analysis of monoclonal antibodies (mAbs) by a middle-down approach is a growing field that attracts the attention of many researchers and biopharma companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before mass spectrometry (MS) analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light (Lc) and heavy (Hc) chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes [~] 24 minutes --40-60% faster than regular LC-MS/MS analysis -- to acquire fragmentation data using different dissociation methods.

biochemistry↗