Systematic LC-MS Analysis of Labile Post-Translational Modifications in Complex Mixtures

2009 ◽  
Vol 8 (5) ◽  
pp. 2608-2614 ◽  
Author(s):  
Christine Carapito ◽  
Clementine Klemm ◽  
Ruedi Aebersold ◽  
Bruno Domon
Keyword(s):  
Complex Mixtures ◽  
Post Translational Modifications ◽  
Ms Analysis

scholarly journals 1H quantitative NMR and UHPLC-MS analysis of seized MDMA/NPS mixtures and tablets from night-club venues

2019 ◽  
Vol 11 (37) ◽  
pp. 4795-4807 ◽  
Author(s):  
Husain A. Naqi ◽  
Stephen M. Husbands ◽  
Ian S. Blagbrough
Keyword(s):  
Complex Mixtures ◽  
Quantitative Nmr ◽  
Ms Analysis ◽  
Night Club

A fast (15 min), accurate and reproducible 1H q-NMR (in D2O) analysis performed on many samples containing complex mixtures of MDMA and NPS revealing significant differences in their ratios.

One-shot LC–MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and β-crystallins induced by γ-irradiation

Amino Acids ◽  
2016 ◽  
Vol 48 (12) ◽  
pp. 2855-2866 ◽  
Author(s):  
Ingu Kim ◽  
Takeshi Saito ◽  
Norihiko Fujii ◽  
Takashi Kanamoto ◽  
Noriko Fujii
Keyword(s):  
Γ Irradiation ◽  
Post Translational Modifications ◽  
Ms Analysis

scholarly journals UC/MALDI-MS analysis of HDL; evidence for density-dependent post-translational modifications

2007 ◽  
Vol 268 (2-3) ◽  
pp. 227-233 ◽  
Author(s):  
Jeffery D. Johnson ◽  
Ronald R. Henriquez ◽  
Shane E. Tichy ◽  
David H. Russell ◽  
Catherine J. McNeal ◽  
...  
Keyword(s):  
Maldi Ms ◽  
Post Translational Modifications ◽  
Density Dependent ◽  
Ms Analysis

scholarly journals Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

2020 ◽  
Vol 412 (25) ◽  
pp. 6833-6848
Author(s):  
Silvia Millán-Martín ◽  
Craig Jakes ◽  
Sara Carillo ◽  
Tom Buchanan ◽  
Marc Guender ◽  
...  
Keyword(s):  
Product Quality ◽  
Magnetic Beads ◽  
Peptide Mapping ◽  
Analysis Data ◽  
Quality Attributes ◽  
Post Translational Modifications ◽  
Subsequent Test ◽  
Ms Analysis ◽  
Recombinant Product ◽  
Product Sequence

Abstract Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.

scholarly journals Monitoring of immunoglobulin N- and O-glycosylation in health and disease

Glycobiology ◽  
2019 ◽  
Vol 30 (4) ◽  
pp. 226-240 ◽  
Author(s):  
Noortje de Haan ◽  
David Falck ◽  
Manfred Wuhrer
Keyword(s):  
Current Status ◽  
Specific Information ◽  
Mass Spectrometers ◽  
Post Translational Modifications ◽  
Effector Functions ◽  
Ms Analysis ◽  
Health And Disease ◽  
Tandem Mass Spectrometric ◽  
Robust Separation

AbstractProtein N- and O-glycosylation are well known co- and post-translational modifications of immunoglobulins. Antibody glycosylation on the Fab and Fc portion is known to influence antigen binding and effector functions, respectively. To study associations between antibody glycosylation profiles and (patho) physiological states as well as antibody functionality, advanced technologies and methods are required. In-depth structural characterization of antibody glycosylation usually relies on the separation and tandem mass spectrometric (MS) analysis of released glycans. Protein- and site-specific information, on the other hand, may be obtained by the MS analysis of glycopeptides. With the development of high-resolution mass spectrometers, antibody glycosylation analysis at the intact or middle-up level has gained more interest, providing an integrated view of different post-translational modifications (including glycosylation). Alongside the in-depth methods, there is also great interest in robust, high-throughput techniques for routine glycosylation profiling in biopharma and clinical laboratories. With an emphasis on IgG Fc glycosylation, several highly robust separation-based techniques are employed for this purpose. In this review, we describe recent advances in MS methods, separation techniques and orthogonal approaches for the characterization of immunoglobulin glycosylation in different settings. We put emphasis on the current status and expected developments of antibody glycosylation analysis in biomedical, biopharmaceutical and clinical research.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

2020 ◽  
Vol 477 (7) ◽  
pp. 1219-1225 ◽  
Author(s):  
Nikolai N. Sluchanko
Keyword(s):  
Protein Function ◽  
Intrinsically Disordered Protein ◽  
Structural Basis ◽  
Major Protein ◽  
Post Translational Modifications ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
Biochemical Journal ◽  
Multiple Binding ◽  
And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

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.

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