Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis

Abstract

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be “Switched Off” via Bioorthogonal Chemistry Inside Live Cells

“…Tetrazine ligation has emerged as a powerful bioorthogonal labeling reaction that is distinguished for its speed, selectivity, and tolerance of a wide range of functional groups. − Among their numerous applications in chemical biology, tetrazines have been used as bioorthogonal handles in proteomic workflows. − Recently, the Carrillo group has demonstrated that 3,6-dialkylthiotetrazines can undergo dynamic nucleophilic substitution reactions and that post-functionalization through Diels–Alder reaction with norbornene gives dihydropyridazine products that no longer exchange. , Applications to the syntheses of macrocycles, macromolecular cages, and to polymer chemistry were described, but applications in biology had not been reported . Recently, our group has described a method for preparing thiomethyltetrazines via a one-pot reaction between carboxylic esters and methylthiocarbohydrazide iodide .…”

Expanding Glycomic Investigations through Thiol-Derivatized Glycans

“…Glycomics investigations commonly rely on sophisticated techniques such as hydrophilic interaction chromatography (HILIC), polymeric anion exchange (WAX), and porous graphitic carbon (PGC) to purify complex glycans [ 4 , 5 ] and mass spectrometry (MS) [ 6 ] to detect desired glycan-containing fractions, and it is further challenging to perform chemical modifications of the selected glycans to facilitate biological studies [ 7 ]. In contrast, the field of proteomics has brought about innovative [ 8 ], selective, mild [ 9 ], and occasionally reversible modifications [ 10 ] of proteins enabling site-specific tagging [ 11 ] and pull-down approaches [ 12 ]. Therefore, the field of proteomics has rapidly progressed with assigning structures and functions to an ever-increasing number of targets [ 13 ].…”

Thiomethyltetrazines are Reversible Covalent Cysteine Warheads whose Dynamic Behavior can be "Switched off” via Bioorthogonal Chemistry Inside Live Cells

“…[48][49][50][51] Among their numerous applications in chemical biology, tetrazines have been used as bioorthogonal handles in proteomic workflows. [52][53][54][55][56][57] Recently, the Carrillo group has demonstrated that 3,6-dialkylthiotetrazines can undergo dynamic nucleophilic substitution reactions, and that post-functionalization through Diels-Alder reaction with norbornene gives dihydropyridazine products that no longer exchange. 58,59 Applications to the syntheses of macrocycles, macromolecular cages, and to polymer chemistry were described, but applications in biology had not been reported.…”