Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

Abstract

A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time-consuming steps needed for poststains. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to that of Pro-Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis was performed to confirm the specificity of the newly developed method.

Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative

“…S1) with stronger fluorescence due to -conjugation of pyrene structure compared with dansylhydrazine and 4H-[1]-benzopyrano [4,3-b]thiophene-2carboxylic acid hydrazide was designed and synthesized to detect glycoproteins as a better alternative. For UGF202 prestain, the mechanism of specific capture of glycoproteins is also based on the oxidative reaction of hydroxyl groups on adjacent carbon atoms of carbohydrates to aldehydes by periodic acid (HIO 4 ) as described in our previous research [10,11]. According to the results, as low as 0.5 ng glycoprotein could be selectively visualized by UGF202 pre-stain, which is similar to that of Pro-Q Emerald 300 stain.…”

“…No further steps are required, and gels can be analyzed instantaneously after electrophoresis. In our previous studies, we have developed two glycoprotein pre‐staining methods by using dansylhydrazine and 4H‐[1]‐benzopyrano[4,3‐b]thiophene‐2‐carboxylic acid hydrazide , respectively. Down to 4–8 ng of glycoproteins could be selectively detected within 40 min by these two methods.…”

Multi-omics Frontiers in Algal Research: Techniques and Progress to Explore Biofuels in the Postgenomics World

“…2DE coupled with conventional staining methods and 2DE analysis tools empowered comparative study of two biological entities, while the infusion of the technique with MS analysis led to the identification of the differentially modulated proteins. Besides, staining of the 2DE gels with post-translational modification (PTM)-specific stains such as Pro-Q Diamond (phosphoproteins) and Pro-Q Emerald, Dansylhydrazine (glycoproteins) can used to study specific PTMs (Marondedze et al, 2013;Wang et al, 2014). 2DE dominated the proteomics era until the introduction of gelfree techniques.…”

Gel-based proteomics in plants: time to move on from the tradition

“…Over 4000 proteins can be identified using 2D gel electrophoresis combined with MS (Imin et al, 2001). PTM specific stains have been developed for phosphoproteins (e.g., Pro-Q Diamond) and glycoproteins (e.g., Pro-Q Emerald, Dansylhydrazine), both of which have emerged as good techniques to study specific PTMs using 2D gels (Marondedze et al, 2013; Wang et al, 2014). However, 2D gels have limitations in terms of separation resolution of complex proteomes, number of spots and protein recovery; all which can be improved by merging methods developed separately for each step in the gel-based proteomic work flow.…”