Posttranslational Modifications of Intact Proteins Detected by NMR Spectroscopy: Application to Glycosylation

Abstract

Posttranslational modifications (PTMs) are an integral part of the majority of proteins.T he characterization of structure and function of PTMs can be very challenging especially for glycans.E xisting methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications,t he inability to identify linkage types and thus chemical structure.W epresent adirect, robust, and simple NMR spectroscopym ethod for the detection and identification of PTMs in proteins.N oi sotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approachiswell suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.

Human plasma protein N-glycosylation

“…These findings have resulted in an increasing interest in the glycosylation analysis of easily accessible biofluids such as plasma, and many correlations have been established between disease (states) and the abundance of specific glycosylation traits. Recent advances in sample preparation methods, separation techniques, mass spectrometry, and the development of robotized platforms are easing routine analysis of the total plasma N -glycome and allow the screening of large cohorts in reasonable times, thus enhancing the possibilities to search for putative biomarkers and predictions tools [ 38 , 109 , 390 – 392 ]. This, however, requires that the observed differences in glycosylation can be interpreted in a biologically meaningful manner, and traced back to changes in the levels of glycosylated proteins, and to possible glycosylation changes of specific proteins.…”

“…Consequently, different levels of detail are available with respect to the glycosylation of the major glycoproteins that make up human plasma. Analysis methods encountered in this review are very diverse, including NMR, lectin capturing, LC-fluorescence, as well as several mass spectrometric methods [ 72 , 390 , 393 ]. NMR has proven definitive for providing structural features of a glycan, but is limited by sample throughput and amount of material needed, as well as by its inability to characterize multiple species present in a complex sample [ 390 ].…”

“…Analysis methods encountered in this review are very diverse, including NMR, lectin capturing, LC-fluorescence, as well as several mass spectrometric methods [ 72 , 390 , 393 ]. NMR has proven definitive for providing structural features of a glycan, but is limited by sample throughput and amount of material needed, as well as by its inability to characterize multiple species present in a complex sample [ 390 ]. Mass spectrometric methods applied in bottom-up studies of glycopeptides generally only provide glycosylation information on a compositional level, but may in addition provide site-specific glycosylation information by revealing the amino acid sequence as well as glycan attachment site [ 182 ].…”

The NMR signature of gluconoylation: a frequent N-terminal modification of isotope-labeled proteins

“…In case of large proteins and severe line broadening, working under denaturing conditions might be an option. Several PTMs have been detected under such conditions with 1 H– 13 C correlations (Schubert et al 2015 ; Grassi et al 2017 ; Peng et al 2018 ). The chemical shifts of the hydrolysis product gluconate might slightly vary, because they depend on the pH and also on the presence of Ca 2+ that can be chelated (Pallagi et al 2010 ), but the gluconoyl chemical shifts should be widely independent of the pH.…”

Detection of methylation, acetylation and glycosylation of protein residues by monitoring13C chemical-shift changes: A quantum-chemical study

“…Since the pioneer observation of lysine methylation of a bacterial protein by Ambler & Rees (1959) , there has been a rising interest in investigating protein post-translational modifications (PTMs) and their role as modulators of protein activity ( Zobel-Thropp, Gary & Clarke, 1998 ; Bienkiewicz & Lumb, 1999 ; Bannister, Schneider & Kouzarides, 2002 ; Paik, Paik & Kim, 2007 ; Bedford & Clarke, 2009 ; Kamieniarz & Schneider, 2009 ; Kamath, Vasavada & Srivastava, 2011 ; Luo, 2012 ; Theillet et al, 2012a ; Theillet et al, 2012b ; Evich et al, 2015 ; Rahimi & Costello, 2015 ; Schubert et al, 2015 ). As a consequence of their relevance, the development of fast and accurate experimental methods for detection of PTMs has also been an object of active research in the field ( Kamath, Vasavada & Srivastava, 2011 ).…”