Site-Specific Phosphorylation Profiling ofArabidopsisProteins by Mass Spectrometry and Peptide Chip Analysis

2008 ◽  
Vol 7 (6) ◽  
pp. 2458-2470 ◽  
Author(s):  
Sergio de la Fuente van Bentem ◽  
Dorothea Anrather ◽  
Ilse Dohnal ◽  
Elisabeth Roitinger ◽  
Edina Csaszar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Site Specific ◽  
Peptide Chip ◽  
Chip Analysis

The challenge of detecting modifications on proteins

2020 ◽  
Vol 64 (1) ◽  
pp. 135-153 ◽  
Author(s):  
Lauren Elizabeth Smith ◽  
Adelina Rogowska-Wrzesinska
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Chemical Properties ◽  
Lysine Acetylation ◽  
Mass Determination ◽  
Post Translational Modifications ◽  
Site Specific ◽  
The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

scholarly journals State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis

2021 ◽  
Vol 14 (6) ◽  
pp. 498
Author(s):  
Evolène Deslignière ◽  
Anthony Ehkirch ◽  
Bastiaan L. Duivelshof ◽  
Hanna Toftevall ◽  
Jonathan Sjögren ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Phase ◽  
Ion Mobility ◽  
State Of The Art ◽  
Native Mass Spectrometry ◽  
Site Specific ◽  
Drug Conjugates ◽  
Conjugation Process ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry

2018 ◽  
Vol 17 (4) ◽  
pp. 1654-1663 ◽  
Author(s):  
Lennard J. M. Dekker ◽  
Lona Zeneyedpour ◽  
Sandor Snoeijers ◽  
Jos Joore ◽  
Sieger Leenstra ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Site Specific

scholarly journals Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

2019 ◽  
Vol 38 (4-5) ◽  
pp. 356-379 ◽  
Author(s):  
Haopeng Xiao ◽  
Fangxu Sun ◽  
Suttipong Suttapitugsakul ◽  
Ronghu Wu
Keyword(s):  
Mass Spectrometry ◽  
Biological Systems ◽  
Protein Glycosylation ◽  
Site Specific ◽  
Specific Analysis
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