FluoroTRAQ: Quantitative Analysis of Protein S-Nitrosylation through Fluorous Solid-Phase Extraction Combining with iTRAQ by Mass Spectrometry

Abstract

S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.

Application of nanomaterials in proteomics-driven precision medicine

“…Subsequently, the fluorescence-tagged SNO-peptides were captured by nanographite fluoride specifically through fluorous-fluorous interactions. Taking advantage of the highly fluorinated level and the high surface area of nanographite fluoride, the enrichment approach was shown to have remarkable selectivity, good sensitivity, high post-enrichment recovery, and large enrichment capacity 42 , 43 .…”

Protein S-Nitrosation: Biochemistry, Identification, Molecular Mechanisms, and Therapeutic Applications

“…FluoroTRAQ combines the fluorous tag switch technique, fluorous solid-phase extraction of SNO–peptides, and iTRAQ . Reduced S -nitrosothiols are labeled with isotopic N -[(3-perfluorooctyl)­propyl]­iodoacetamide, allowing enrichment and quantification of the SNO proteome (Figure ).…”

“…FluoroTRAQ combines the fluorous tag switch technique, fluorous solid-phase extraction of SNO−peptides, and iTRAQ. 80 Reduced S-nitrosothiols are labeled with isotopic N-[(3-perfluorooctyl)propyl]iodoacetamide, allowing enrichment and quantification of the SNO proteome (Figure 3). After protease digestion, fluorouslabeled peptides are enriched by fluorous solid-phase extraction, which utilizes the fluorous solid phase to easily separate fluorous from nonfluorous molecules.…”

FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions

“…As a result, only several hundred S-nitrosylated proteins could be identified by the biotin-switch or iodo-TMT methods in plants 23 (see the summary Supplementary Table 1 ), limiting the deeper profiling of S-nitrosylation. Several other labeling reagents, such as isotope coded affinity tag (ICAT) 25 , iodo-TMT 26 , 27 , cysteine-specific phosphonate adaptable tag (CysPAT) 28 , bioorthogonal cleavable-linker (Cys-BOOST) 29 , and fluorous affinity tag (FAT) 30 , were also used to label and enrich S-nitrosopeptides. However, these commonly practiced methods were rarely applied to plants.…”

“…Among these reagents, FAT with covalent C-F bonds avoids unexpected dissociation during the MS process and reduces the complexity of tandem MS, allowing the identification of low abundance targets 30 . FAT has been successfully applied in some proteomic studies of post-translational modification, such as protein phosphorylation 31 , tyrosine nitration 32 , HNE modification 33 , and S-nitrosylation 30 . However, whether FATs are applicable in plants to acquire in-depth S-nitrosylation proteomic analysis requires further testing.…”