Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.     

Protein staining influences the quality of mass spectra obtained by peptide mass fingerprinting after separation on 2-d gels. A comparison of staining with coomassie brilliant blue and sypro ruby

When separating protein mixtures on 2-D gels for proteomics purposes, fluorescent staining is superior in sensitivity and linear response as compared to Coomassie Brilliant Blue (CBB) and silver staining, respectively. We have compared the quality of mass spectra for proteins obtained from gels stained with CBB and SYPRO Ruby (SR) and found significant differences. These differences can be seen both in inferior signal/noise ratios and number of peptides detected with the fluorescent stain.     

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

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.     

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.Gel electrophoresis is an accessible, widely applicable and mature protein resolving technology. As the original top-down approach to proteomic analyses, among its many attributes the high resolution achievable by two dimensional gel-electrophoresis (2DE)¹ ensures that it remains an effective analytical technology despite the appearance of alternatives. However, in-gel detection remains a limiting factor for gel-based analyses; available technology generally permits the detection and quantification of only relatively abundant proteins (35). Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques. Pre- and post-fractionation technologies have been developed to address this central issue in proteomics but these are not without limitations (1–5). Thus improved detection methods for gel-based proteomics continue to be a high priority, and the literature is rich with different in-gel detection methods and innovative improvements (6–34). This history of iterative refinement presents a wealth of choices when selecting a detection strategy for a gel-based proteomic analysis (35).Perhaps the best known in-gel detection method is the ubiquitous Coomassie Blue (CB) stain; CB has served as a gel stain and protein quantification reagent for over 40 years. Though affordable, robust, easy to use, and compatible with mass spectrometry (MS), CB staining is relatively insensitive. In traditional organic solvent formulations, CB detects ∼ 10 ng of protein in-gel, and some reports suggest poorer sensitivity (27, 29, 36, 37). Sensitivity is hampered by relatively high background staining because of nonspecific retention of dye within the gel matrix (32, 36, 38, 39). The development of colloidal CB (CCB) formulations largely addressed these limitations (12); the concentration of soluble CB was carefully controlled by sequestering the majority of the dye into colloidal particles, mediated by pH, solvent, and the ionic strength of the solution. Minimizing soluble dye concentration and penetration of the gel matrix mitigated background staining, and the introduction of phosphoric acid into the staining reagent enhanced dye-protein interactions (8, 12, 40), contributing to an in-gel staining sensitivity of 5–10 ng protein, with some formulations reportedly yielding sensitivities of 0.1–1 ng (8, 12, 22, 39, 41, 42). Thus CCB achieved higher sensitivity than traditional CB staining, yet maintained all the advantages of the latter, including low cost and compatibility with existing densitometric detection instruments and MS. Although surpassed by newer methods, the practical advantages of CCB ensure that it remains one of the most common gel stains in use.Fluorescent stains have become the routine and sensitive alternative to visible dyes. Among these, the ruthenium-organometallic family of dyes have been widely applied and the most commercially well-known is Sypro Ruby (SR), which is purported to interact noncovalently with primary amines in proteins (15, 18, 19, 43). Chief among the attributes of these dyes is their high sensitivity. In-gel detection limits of < 1 ng for some proteins have been reported for SR (6, 9, 14, 44, 45). Moreover, SR staining has been reported to yield a greater linear dynamic range (LDR), and reduced interprotein variability (IPV) compared with CCB and silver stains (15, 19, 46–49). SR is easy to use, fully MS compatible, and relatively forgiving of variations in initial conditions (6, 15). The chief consequence of these advances remains high cost; SR and related stains are notoriously expensive, and beyond the budget of many laboratories. Furthermore, despite some small cost advantage relative to SR, none of the available alternatives has been consistently and quantitatively demonstrated to substantially improve on the performance of SR under practical conditions (9, 50).Notably, there is evidence to suggest that CCB staining is not fundamentally insensitive, but rather that its sensitivity has been limited by traditional densitometric detection (50, 51). When excited in the near IR at ∼650 nm, protein-bound CB in-gel emits light in the range of 700–800 nm. Until recently, the lack of low-cost, widely available and sufficiently sensitive infrared (IR)-capable imaging instruments prevented mainstream adoption of in-gel CB infrared fluorescence detection (IRFD); advances in imaging technology are now making such instruments far more accessible. Initial reports suggested that IRFD of CB-stained gels provided greater sensitivity than traditional densitometric detection (50, 51). Using CB R250, in-gel IRFD was reported to detect as little as 2 ng of protein in-gel, with a LDR of about an order of magnitude (2 to 20 ng, or 10 to 100 ng in separate gels), beyond which the fluorescent response saturated into the μg range (51). Using the G250 dye variant, it was determined that CB-IRFD of 2D gels detected ∼3 times as many proteins as densitometric imaging, and a comparable number of proteins as seen by SR (50). This study also concluded that CB-IRFD yielded a significantly higher signal to background ratio (S/BG) than SR, providing initial evidence that CB-IRFD may be superior to SR in some aspects of stain performance (50).Despite this initial evidence of the viability of CB-IRF as an in-gel protein detection method, a detailed characterization of this technology has not yet been reported. Here a more thorough, quantitative characterization of CB-IRFD is described, establishing its lowest limit of detection (LLD), IPV, and LDR in comparison to SR. Finally a wealth of modifications and enhancements of CCB formulations have been reported (8, 12, 21, 24, 26, 29, 40, 41, 52–54), and likewise there are many commercially available CCB stain formulations. To date, none of these formulations have been compared quantitatively in terms of their relative performance when detected using IRF. As a general detection method for gel-based proteomics, CB-IRFD was found to provide comparable or even slightly superior performance to SR according to most criteria, including sensitivity and selectivity (50). Furthermore, in terms of LDR, CB-IRFD showed distinct advantages over SR. However, assessing proteomes resolved by 2DE revealed critical distinctions between CB-IRFD and SR in terms of protein quantification versus threshold detection: neither stain could be considered unequivocally superior to the other by all criteria. Nonetheless, IRFD proved the most sensitive method of detecting CB-stained protein in-gel, enabling high sensitivity detection without the need for expensive reagents or even commercial formulations. Overall, CB-IRFD is a viable alternative to SR and other mainstream fluorescent stains, mitigating the high cost of large-scale gel-based proteomic analyses, making high sensitivity gel-based proteomics accessible to all labs. With improvements to CB formulations and/or image acquisition instruments, the performance of this detection technology may be further enhanced.     

Proteomic capacity of recent fluorescent dyes for protein staining

Staining of two-dimensional gel constitutes a crucial step in comparative proteome analysis with respect to both the number of proteins analysed, the accuracy of spot quantification and reproducibility. In this work, we compared the efficiency of recent fluorophores to stain Arabidopsis total protein extract: Sypro Ruby (SR), Deep Purple (DP) and 5-hexadecanoylamino-fluorescein (C16-F). In addition, classical visible dyes, colloidal Coomassie blue (CCB) and silver nitrate (SN), were also included. High quality images were obtained for the three fluorescent dyes, DP giving the cleaner background, whereas spikes were observed with SR and a rough background with C16-F. On the other hand, saturation occurred for abundant spots with SR and DP. For a same protein load the number of detected spots ranged between 250 for CCB and 800 for SR in the sequence SR > DP approximately SN > C16-F > CCB. These differences were shown to rely mainly on the sensitivity between dyes leading to the detection of additional spots belonging to classes of lower abundance. Analysis of the distribution of variation coefficients for spots from replicates showed differences in the staining reproducibility between dyes that ranged in the order SR > C16-F > DP > SN > CCB. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.     

Identification of Endothelial Proteins by MALDI-MS Using a Compact Disc Microfluidic System

Vascular endothelial proteins have been analyzed using two-dimensional (2D) gel electrophoresis and subsequent mass spectrometry, with separate methods for the intervening sample preparations. Compact disc (CD) technology was found to be rapid, giving high overall yield both with ordinary Coomassie staining and with Sypro Ruby staining. Combined with automatic in-gel digestion, the CD technology has great capacity for large numbers of protein analysis, although for limited sample numbers, manual methods can give similar sequence coverage. In a test set of 48 samples, 45 proteins were identified using the CD preparation technique, 32 identified with higher sequence coverage using the CD technique, 7 with higher using ZipTips in a robotic workstation, and 5 with higher coverage using dried droplets of unpurified samples. In the process of these methodological comparisons, basic patterns for 116 endothelial proteins were defined, representing 297 separate protein spots on the 2D gels.     

A sensitive method for detection of proteins in polyacrylamide gels and on protein blots using acidic henna leaf extract

Summary In the present communication we describe a process for the preparation of an acidic henna leaf extract useful as a general protein stain for both polyacrylamide gels and protein blots. The staining is reversible by changing its pH and does not require protein fixation or destaining steps. This staining procedure is simpler and also, its sensitivity is comparable with the two commonly used organic stains i.e. Coomassie Blue and Amido Black. Under optimum conditions the protein detection limits of acidic henna leaf extract varied from 50 ng to 100 ng.     

Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue

A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research.     

High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Comparison of SYPRO Ruby and Deep Purple using commonly available UV transilluminator: wide-scale application in proteomic research

Fluorescent stains are becoming increasingly useful in proteomics research involving protein expression as well as post-translational modification studies and are particularly useful for samples which are expensive and scarce. The fluorescent dyes Deep Purple and SYPRO Ruby are widely used in protein expression studies. Using UV transillumination and Charged Coupled Device (CCD) based imaging system, their relative sensitivity to detect proteins separated by two-dimensional polyacrylamide gel electrophoresis and downstream protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was compared. Using mouse liver homogenate, we detected a greater number of spots using SYPRO Ruby over Deep Purple stain. However, the number of matched peptides and the percentage of amino acid residues identified for 21 different proteins were comparable suggesting their equivalency for LC-MS/MS identification. In spite of comparable MS compatibility, we recommend the use of SYPRO Ruby for expression proteomics due to its higher sensitivity in detecting protein spots.     

Comparison of SYPRO Ruby and Flamingo fluorescent stains for application in proteomic research

Fluorescent dyes are widely used for the detection and quantitation of proteins separated by polyacrylamide gel electrophoresis. SYPRO Ruby is one such fluorescent dye widely used for this purpose. More recently, another fluorescent dye, Flamingo, is available for expression proteomic research. Using a standard ultraviolet (UV) transilluminator and a charge-coupled device (CCD)-based imaging system, the relative sensitivity of these two different fluorescent stains with regard to detection of protein spots separated by two-dimensional gel electrophoresis (2D-GE) and identification by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) were compared. Using mouse kidney and liver homogenates as well as Escherichia coli extract, we detected a greater number of protein spots using Flamingo compared with SYPRO Ruby. In addition, when we compared the number of matched peptides and the percentage of amino acid residues identified for 22 different protein spots of mouse kidney proteome, we observed a higher number of matched peptides and a higher percentage of amino acid residues for the majority of the proteins using Flamingo compared with SYPRO Ruby. Also, we were able to characterize a protein spot that can be detected by Flamingo only. Therefore, we recommend Flamingo over SYPRO Ruby to be used for studies on expression proteomics.     

Quantitation of chick tissue zinc-metallothionein by gel electrophoresis and silver stain enhancement

Discontinuous gradient gel electrophoretic systems were developed to quantitate zinc-metallothionein (Zn-MT) in chick tissues, liver and pancreas. Gels were stained with Coomassie Blue initially, then enhanced by silver stain. At least 4 micrograms of Zn-MT could be detected after Coomassie Blue stain, and 1 microgram Zn-MT detected following silver stain enhancement. Significant linearity (correlation coefficient = 0.99) of a standard curve was established in the Coomassie Blue stained gels. The results of our experiment suggest that electrophoretic analysis is a simple and feasible method for the quantitation and identification of Zn-MT in chick tissues.     

Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway

Differentiated melanocytic cells produce melanin, through several redox reactions including tyrosinase-catalyzed DOPA oxidation to DOPA quinone. We now developed a method based on DOPA oxidase in-gel detection and Sypro Ruby fluorometric normalization to investigate induction of specific DOPA oxidase isoforms in response to hydrogen peroxide-mediated stress, and to ask whether this is associated with p53-dependent adaptive responses. This report shows that hydrogen peroxide leads to comparable induction of 60 and 55 kDa DOPA oxidases in poorly pigmented B16 melanoma, in contrast to sole induction of a major 55 kDa DOPA oxidase in their highly pigmented counterparts. In the latter cells, this response also increases p53 concomitant with joint induction of p53-activated proteins like the cell-cycle inhibitor p21WAF1 and pro-apoptotic bax, with no comparable effect on expression of anti-apoptotic bcl-2. Together, these data suggest that response to hydrogen peroxide involves p53-mediated growth-restrictive signaling and unequal induction of specific DOPA oxidases in melanocytic cells with unequal basal pigmentation.     

Isolation of a component from commercial coomassie brilliant blue R-250 that stains rubrophilin and other proteins red on polyacrylamide gels

Commercially available Coomassie Brilliant Blue R-250 (C.I. 42660) is a popular and useful dye that stains most proteins blue on polyacrylamide gels. Some proteins from brain (rubrophilin), collagens, histones and parotid gland proteins are distinctly red when stained with Coomassie Blue. Commonly used Coomassie Brilliant Blue R-250 preparations may contain more than 30 distinct colored and fluorescent components that can be separated on silica gel chromatographic columns. A specific component has been isolated on silica gel columns that stains rubrophilin and other proline-rich proteins a reddish color. Fast atom bombardment mass spectrometry of the isolated rubrophilin staining principle indicates a molecular weight of 634 as compared to 826 for the major dye in the original Coomassie Brilliant Blue R-250. Infrared spectrometry is consistent with a difference between the rubrophilin staining principle and Coomassie Brilliant Blue R-250 of a toluene sulfonic acid residue.     

A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis

Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry.     

A fluorescence-based Coomassie Blue protocol for two-dimensional gel-based proteomics

A sensitive and convenient “visible SYPRO” staining protocol was developed for visualizing proteins after SDS-PAGE. Gels were sensitized with SYPRO Ruby and then stained with the Coomassie Brilliant Blue G-250 protocol (Blue Silver). This combined protocol had similar or better linearity than staining with only SYPRO Ruby or Blue Silver, respectively. In addition, this method was more sensitive than that of Blue Silver, simpler than that of SYPRO Ruby, and compatible with subsequent mass spectrometry analysis.     

Mass spectral compatibility of four proteomics stains

With the recent introduction of new fluorescence stains to the proteomics market, there is now more choice available. SYPRO Ruby, LavaPurple, Flamingo, and Krypton total protein stains were compared for ease of use, image quality, and compatibility with protein identification by peptide mass fingerprinting (PMF) (MALDI-TOF). All four stains produced good images but with slightly different staining patterns. SYPRO was found to inhibit identification of cysteine and tryptophan containing peptides, which reduced protein identification.     

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.