A rapid and simple 8‐quinolinol‐based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS‐PAGE, a fluorescent detection method named 8‐Quinolinol (8‐Q) stain is described. 8‐Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al³⁺) to the phosphate groups on the proteins and the metal chelating property of 8‐Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4～8 ng of α‐casein and β‐casein, 16～32 ng of ovalbumin and κ‐casein which is more sensitive than Stains‐All but less sensitive than Pro‐Q Diamond. The protocol of 8‐Q requires only 70 min in 0.75 mm mini‐size or 1.0 mm large‐size gels with five changes of solutions without destaining step; Pro‐Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC‐MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8‐Q could be an alternative to simplify the analytical operations for phosphoproteomics research.     

Sensitive phosphoprotein detection in SDS‐PAGE via Anthracene Chrome Red A stain

Protein phosphorylation, one of the most important post‐translational modifications, plays critical roles in many biological processes. Thus, it is necessary to precisely detect, identify and understand the phosphoproteins from protein mixture for the study of cell biology. We introduce a sensitive and specific detection method for phosphoproteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Anthracene Chrome Red A (ACRA) combined with the trivalent metal ion (Al³⁺) is converted to fluorescent complex and the fluorescence is sharply increased by a change of pH environment. Phosphoproteins and non‐phosphoproteins can be easily distinguished by the fluorescence quenching due to the structural change of ACRA‐Al³⁺‐phosphoprotein complex, unlike non‐phosphoprotein complex. The method using ACRA is a negative staining based on the fluorescence quenching and has a high sensitivity comparable to Pro‐Q Diamond stain. ACRA stain can detect 1–2 ng of α‐casein and β‐casein, 8–16 ng of ovalbumin (OVA) and κ‐casein within 130 min. Moreover, the ACRA stain showed similar linear dynamic ranges and RSD to Pro‐Q stain. The linear dynamic ranges of ACRA and the values of correlation coefficient were for OVA (8–500 ng, correlation coefficient r = 0.999), α‐casein (4–500 ng, r  = 0.992), β‐casein (4–500 ng, r  = 0.996), and κ‐casein (8–500 ng, 0.998), respectively. On the other hand, the values of the relative standard deviations (RSD) ranged from 2.33 to 3.56% for ACRA. The method is sensitive, specific, simple, rapid and compatible with total protein stain such as SYPRO Ruby stain. Therefore, ACRA stain can be an advanced method for phosphoprotein detection in gels.     

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS‐PAGE

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS‐PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al³⁺ were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16–32 ng of α‐casein and β‐casein, 62 ng of ovalbumin, phosvitin, and κ‐casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.     

A color‐code for glycosaminoglycans identification by means of polyacrylamide gel electrophoresis stained with the cationic carbocyanine dye Stains‐all

Cationic dyes such as toluidin blue are commonly employed to visualize glycosaminoglycans (GAGs) on electrophoresis gels; however, the carbocyanine‐based dye Stains‐all have been increasingly used to stain the non‐sulfated hyaluronic acid and other GAGs in submicrogram quantities. In this short communication, we demonstrate that Stains‐all is able to stain the most common GAGs on polyacrylamide gels with distinct and contrasting colors in a reproducible manner. We also show that this staining method is useful to identify GAGs present both in mixtures and in submicrogram quantities. Therefore, Stains‐all has shown to be useful in identifying GAGs on polyacrylamide gels with basis on their specific colors, at least on screening level.     

Alternative visualization of SDS‐PAGE separated phosphoproteins by alizarin red S‐aluminum (III)‐appended complex

A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S‐aluminum (III)‐appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS‐PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α‐ (seven or eight phosphates) and β‐casein (five phosphates), 125 ng of ovalbumin (two phosphates), and κ‐casein (one phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125–4000 ng range. As a result, alizarin red S‐aluminum (III) stain may provide a new choice for selective, economic, and convenient visualization of phosphoproteins.     

A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid

A fluorescent quenching detection method for phosphoproteins in SDS‐PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al³⁺ in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA‐Al³⁺‐phosphoprotein appended complex. Approximately 4–8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS‐PAGE), which is similar to that of the most commonly used Pro‐Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC‐MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel‐separated phosphoproteins.     

Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision‐induced dissociation top‐down approach for characterizing phosphoproteins on a quadrupole time‐of‐flight mass spectrometer. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36⁺ were detected. Tandem mass spectrometry was performed on β‐casein ions of different charge states (12⁺, and 15⁺ to 28⁺) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N‐terminal amide bond adjacent to proline residues (Xxx‐Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx‐Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β‐casein further complicated the spectra. Our results suggest that when interpreting top‐down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.     

RAMA casein zymography: Time‐saving and highly sensitive casein zymography for MMP7 and trypsin

To detect metalloproteinase‐7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high‐sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100‐fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin.     

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

A simple and sensitive fluorescent staining method for the detection of proteins in SDS‐PAGE, namely IB (improved 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid) stain, is described. Non‐covalent hydrophobic probe 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non‐specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.     

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.     

Two‐step Immobilized Metal Affinity Chromatography (IMAC) for Phosphoproteomics Using Mass Spectrometry

In this study, a new strategy named two‐step IMAC is demonstrated as a novel prelude to MS analysis of phosphoproteome by increasing the enrichment factor of phosphoproteins/phosphopeptides from a protein mixture. In this method, the first IMAC was performed at the protein level to extract the minute amount of phosphoproteins present in the sample. During this step, nonphosphoproteins and other undesired chemicals or inhibitors were excluded. After tryptic digestion, the second IMAC was performed at the peptide level to enrich phosphopeptides present in the tryptic digest, and the eluent from the second IMAC was analyzed by MALDI‐MS. It is particularly noticeable that the eluent from the first IMAC can be directly digested by trypsin without buffer exchange. Our results revealed that β‐casein that was spiked in a protein mixture can be successfully extracted by the first IMAC at a concentration of less than 1–3%, and the two phosphopeptides of β‐casein with single and four phosphorylation sites, respectively, can be captured by the second IMAC. It was found that the two‐step IMAC method could significantly reduce non‐specific bindings from unwanted proteins and greatly enhance the MALDI‐MS signal of phosphopeptide ions compared to the typical one‐step IMAC, by which only IMAC at the peptide level was performed. Two‐step IMAC was also found to tolerate a greater amount and a greater concentration range of proteins than one‐step IMAC, which is especially important when analyzing complicated unknown samples. Furthermore, the MS signal of phosphopeptide ions did not appear to be degraded by the presence of biological matrixes, such as the cell lysate in which the β‐casein was spiked in.     

Facile synthesis of Ti4+‐immobilized affinity silica nanoparticles for the highly selective enrichment of intact phosphoproteins

Currently, great challenges to top‐down phosphoproteomics lie in the selective enrichment of intact phosphoproteins from complex biological samples. Herein, we developed a facile approach for synthesis of Ti⁴⁺‐immobilized affinity silica nanoparticles and applied them to the selective separation and enrichment of intact phosphoproteins based upon the principle of metal(IV) phosphate/phosphonate chemistry. The as‐prepared affinity materials exhibited high selectivity and adsorption capacities for model phosphoproteins (328.9 mg/g for β‐casein, 280.5 mg/g for ovalbumin, and 225.8 mg/g for α‐casein), compared with nonphosphoproteins (79.28 mg/g for horseradish peroxidase, 72.70 mg/g for BSA, and 27.28 mg/g for lysozyme). In addition, the resuability of the affinity silica nanoparticles was evaluated, and the results demonstrated a less than 10% loss of adsorption capacity after six adsorption–regeneration cycles. The practicability of the affinity materials was demonstrated by separating phosphoproteins from protein mixtures and drinking milk samples, and the satisfactory results indicated its potential in phosphoproteomics analysis.     

Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.     

Improved dynamic range of protein quantification in silver‐stained gels by modelling gel images over time

Silver staining is a commonly used protein stain to visualise proteins separated by 2‐DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high‐ and low‐abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end‐point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2‐DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.     

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

A negative detection method for proteins on SDS‐PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water‐soluble protein–dye complex. Negative staining of proteins using EY, allows high‐sensitivity, low‐cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole‐zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high‐throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.     

Facile preparation of titanium phosphate‐modified chitosan for selective capture of phosphopeptides

A facile two‐step method for preparing chitosan‐based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan‐based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α‐casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum‐loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α‐casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α‐casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt‐stressed Arabidopsis thaliana leaves.     

Phosphoproteome profile of human liver Chang's cell based on 2-DE with fluorescence staining and MALDI-TOF/TOF-MS

Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro-Q Diamond dye, for the high-throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2-DE, then sequentially stained with Pro-Q Diamond and Coomassie Blue G-250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI-TOF/TOF-MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2-DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection.     

Background-free,fast protein staining in sodium dodecyl sulfate polyacrylamide gel using counterion dyes,zincon and ethyl violet

A background-free, fast protein staining method in polyacrylamide gel electrophoresis using an acidic dye, zincon (ZC) and a basic dye, ethyl violet (EV) is described. It is based on the counterion dye staining technique that employs two oppositely charged dyes to form an ion-pair complex in staining solution. The selective binding of free dye molecules to proteins in acidic solution produces bluish violet-colored bands. It is a rapid and end-point staining procedure, involving only fixing and staining steps that are completed in 1-1.5 h. The detection limit of this method is 8-15 ng of protein that is comparable to the sensitivity of the colloidal Coomassie Brilliant Blue G (CBBG) stain. Due to its sensitivity and speed, this stain may be more practical than any other dye-based stains for routine laboratory purposes.     

Improved conditions for periodate/Schiff's base‐based fluorescent staining of glycoproteins with dansylhydrazine in SDS‐PAGE

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS‐PAGE was described. Down to 4–8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16‐fold higher than that of original protocol, but similar to that of Pro‐Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel‐separated glycoproteins.     

Fluorogenic Ag+–Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining

Silver staining, which exploits the special bioaffinity and the chromogenic reduction of silver ions, is an indispensable visualization method in biology. It is a most popular method for in‐gel protein detection. However, it is limited by run‐to‐run variability, background staining, inability for protein quantification, and limited compatibility with mass spectroscopic (MS) analysis; limitations that are largely attributed to the tricky chromogenic visualization. Herein, we reported a novel water‐soluble fluorogenic Ag⁺ probe, the sensing mechanism of which is based on an aggregation‐induced emission (AIE) process driven by tetrazolate‐Ag⁺ interactions. The fluorogenic sensing can substitute the chromogenic reaction, leading to a new fluorescence silver staining method. This new staining method offers sensitive detection of total proteins in polyacrylamide gels with a broad linear dynamic range and robust operations that rival the silver nitrate stain and the best fluorescent stains.