Saturation fluorescence labeling of proteins for proteomic analyses

Abstract

We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed.We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells.

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…Focusing on thiols also allows the pIs of labeled proteins to be maintained since the dyes are neutrally charged. Recently, it has been demonstrated that saturation labeling of thiols can also be successfully carried out using the BODIPY dyes (FL-N-(2-aminoethyl) maleimide; FL C1-idoacetamide) by optimizing the labeling reaction conditions [148]. The BODIPY dyes demonstrate a LLD of 10 fmol (without a reported error) and a LDR of three orders of magnitude for yeast enolase I. Inter-protein variation was not referred to but can be inferred from the thiol specific nature of these dyes [148].…”

Role of Peroxiredoxin 1 and Peroxiredoxin 4 in Protection of Respiratory Syncytial Virus-Induced Cysteinyl Oxidation of Nuclear Cytoskeletal Proteins

“…A549 nuclear protein was labeled with BODIPY (Invitrogen) before IEF/ 2-DE according to our previously published protocol (23). To label proteins, 150 to 200 g protein were denatured in 200 l of reaction buffer (50 mM Tris-HCl [pH 7.5], 7 M urea, 2 M thiourea, 2% CHAPS) and incubated with 60-fold excess BODIPY over protein thiols for 2 h at 22°C.…”

“…However, the nuclear proteins that are protected are unknown. To determine their identities, we adapted a sensitive cysteinyl-reactive saturation fluorescence labeling technique to identify proteins that are oxidized in response to RSV in the presence or absence of Prdx knockdown (23). This technique uses BODIPY to covalently label protein cysteines when they are reduced.…”

ProteinS-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment

“…However, IAM is more selective for the thiolate anion and results in lower nonspecific labeling compared with NEM, making IAM-DES a highly specific and selective labeling reagent for the reactive thiols generated after Grx1 reduction. IAM requires a more alkaline pH range (7.5-8.5) for optimum reactivity and reacts more readily with cysteines at their pKa around pH 8.0 (46,71). At higher pH values and temperatures, IAM can alkylate methionines, lysines, arginines, tyrosines, histidines, and the N-terminal and Cterminal carboxyl (20,35,70).…”

“…NEM was added prelysis to avoid thiol oxidation during cell lysis and other processing steps and to avoid loss of protein thiol-GSH mixed disulfides due to transthiolation and disulfide formation during sample preparation (18). NEM is a commonly used thiol-alkylating agent because of its high reactivity and specificity for thiols at physiological pH (6.5-7.5); at higher pH (>7.5), NEM reacts with lysines, arginines, histidines, and tyrosines (37,46) and has been reported to also react with sulfenic acids (49) and possibly with sulfinic acids (29,49). NEM has proven to be a better overall alkylating agent than IAM based on its ability to react with free thiols and sulfenic acids faster, more efficiently, and within a broader effective pH range (49,51).…”