2DE‐based approach for estimation of number of protein species in a cell

Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this article. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting the spots opens a way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or LOD of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, Escherichia coli and Pirococcus furiosus, are 6200 and 3400, respectively. We expect a single human cell (HepG2) to contain minimum 70 000 protein species.     

Normal human mitral valve proteome: A preliminary investigation by gel‐based and gel‐free proteomic approaches

The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre‐MS separation technique based on liquid‐phase IEF and SDS‐PAGE to identify the largest number of proteins. We also demonstrated that some of these proteins, e.g. αB‐Crystallin, septin‐11, four‐and‐a‐half LIM domains protein 1, and dermatopontin, are synthesised by interstitial cells isolated from human mitral valves. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms. Data are available via ProteomeXchange with identifier PXD004397.     

Evaluation of extraction procedures for 2‐DE analysis of aphid proteins

Protein sample preparation is a crucial step in a 2‐DE proteomics approach. In order to establish a routine protocol for the application of proteomics analysis to aphids, this study focuses on the specific protein extraction problems in insect tissues and evaluates four methods to bypass them. The approaches of phenol extraction methanol/ammonium acetate precipitation (PA), TCA/acetone precipitation, PEG precipitation, and no precipitation were evaluated for proteins isolation and purification from apterous adult aphids, Sitobion avenae. For 2‐DE, the PA protocol was optimal, resulting in good IEF and clear spots. PA method yielded the greatest amount of protein and displayed most protein spots in 2‐DE gels, as compared with the TCA/acetone precipitation, PEG precipitation and no precipitation protocols. Analysis of protein yield, image quality and spot numbers demonstrate that the TCA/acetone precipitation protocol is a reproducible and reliable method for extracting proteins from aphids. The PEG precipitation approach is a newly developed protein extraction protocol for aphids, from which more unique protein spots can be detected, especially for detection of acid proteins. These protocols are expected to be applicable to other insects or could be of interest to laboratories involved in insect proteomics, despite the amounts and types of interfering compounds vary considerably in different insects.     

Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysis

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.     

Horizontal comparative fluorescence two‐dimensional gel electrophoresis for improved spot coordinate detection

Vertical comparative 2D fluorescence gel electrophoresis (CoFGE) has recently been shown to increase the reproducibility of coordinate assignment for protein spots, in particular in singular experiments, which cannot be investigated using DIGE. The method applies a standardized marker grid formed by a set of purified proteins to the sample proteome in a conglomerate of 1DE, 2DE, and DIGE. Here, improvements are demonstrated by transferring CoFGE to horizontal 2DE. These include the elimination of the protein modification by residual acrylamide monomer unavoidable in vertical CoFGE, reduced buffer volumes, and highly efficient laboratory procedures. Spot patterns are well defined and can be easily analyzed using commercially available warping algorithms. With horizontal CoFGE also a correction for changes in pI was introduced using a third fluorescent dye. Horizontal CoFGE holds high promises in comparative proteomics.     

An improved pixel-based approach for analyzing images in two-dimensional gel electrophoresis

An improved pixel-based approach for analyzing 2-DE images is presented. The key feature of the method is to create a mask based on all gels in the experiment using image morphology, followed by multivariate analysis on the pixel level. The method reduces the impact of noise and background by identifying regions in the image where protein spots are present, but make no assumption on individual spot boundaries for isolated spots. This makes it possible to detect significant changes in complex regions, and visualize these changes over multiple gels in an easy way. False missing values and spot volumes caused by imposing erroneous spot boundaries are thus circumvented. The approach presented gives improved pixel-based information from the gels, and is also an alternative to existing methods for data-reduction, significance testing and visualization of 2-DE data. Results are compared with software using a common spot boundary approach on an experiment consisting of 35 full size gel images. Gel alignment is required before analysis.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE

Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues.     

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 MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification

The availability of easy-to-handle, sensitive, and cost-effective protein staining protocols for 2-DE, in conjunction with a high compatibility for subsequent MS analysis, is still a prerequisite for successful proteome research. In this article we describe a quick and easy-to-use methodological protocol based on sensitive, homogeneous, and MS-compatible silver nitrate protein staining, in combination with an in-gel digestion, employing the Millipore 96-well ZipPlate system for peptide preparation. The improved quality and MS compatibility of the generated protein digests, as compared to the otherwise weakly MS-compatible silver nitrate staining, were evaluated on real tissue samples by analyzing 192 Coomassie-stained protein spots against their counterparts from a silver-stained 2-DE gel. Furthermore, the applicability of the experimental setup was evaluated and demonstrated by the analysis of a large-scale MALDI-TOF MS experiment, in which we analyzed an additional ~1000 protein spots from 2-DE gels from mouse liver and mouse brain tissue.     

Comparative two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) of human milk to identify dysregulated proteins in breast cancer

Breast cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC‐associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot proteomics study, using one‐dimensional polyacrylamide gel electrophoresis (1D‐PAGE) and mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her milk. Statistically different gel spots were picked for protein digestion followed by nanoliquid chromatography tandem MS (nanoLC‐MS/MS) analysis. The upregulated proteins in BC versus control are alpha‐amylase, gelsolin isoform a precursor, alpha‐2‐glycoprotein 1 zinc isoform CRA_b partial, apoptosis‐inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease. MS data is available via ProteomeXchange with identifier PXD009860.     

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: A gel-free multidimensional electrophoresis approach for proteomic profiling—Exemplified on human follicular fluid

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte–wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.     

Searching for serum tumor markers for colorectal cancer using a 2‐D DIGE approach

Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2‐D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2‐D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down‐regulated in patients (with ratios from ?1.23 to ?10.21) (t‐test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2‐D DIGE results. Thus, 2‐D DIGE approaches show great promise for biomarker discovery in CRC.     

An optimized method to extract poplar leaf proteins for two‐dimensional gel electrophoresis guided by analysis of polysaccharides and phenolic compounds

Commonly used methods for protein extraction from plant leaves, such as extraction with phenol or a combination of trichloroacetic acid and acetone, were ineffective for four tested cultivars of poplar. Moreover, multiple protocols for 2DE of the extracted proteins gave different results when protein profiles of relatively closely related plants were compared. Given that polycyclic compounds strongly hinder 2DE, we analyzed the impact of polyphenols and polysaccharides present in the plant tissues used for protein extraction, on the quality of 2DE protein profiles. Analysis of content of polyphenols and polysaccharides in leaves of poplar cultivars showed that even small differences in concentrations of analyzed metabolites accompany large differences between poplar cultivars when considering the susceptibility of samples to protein extraction for 2DE. High‐quality 2DE results were correlated with decreased amounts of polyphenols. Additional analysis using MS/MS suggested that only levels of total phenolics affected the results of 2DE. Soluble total nonstructural carbohydrates also had a negative effect, but the level of starch was not important. Finally, we present an optimized method for extraction of proteins from poplar leaves, which enables reliable comparative analysis of four different poplar cultivars, that is, “Eridano,” “Villafranca,” “NE‐42,” and “Luisa Avanzo,” which have not yet been used for the proteomic studies.     

Estimation of protein concentration at high sensitivity using SDS‐capillary gel electrophoresis‐laser induced fluorescence detection with 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde protein labeling

3‐(2‐furoyl)quinoline‐2‐carboxaldehyde (FQ) is a sensitive fluorogenic dye, used for derivatization of proteins for SDS‐CGE with LIF detection (SDS‐CGE‐LIF) at silver staining sensitivity (ng/mL). FQ labels proteins at primary amines, found at lysines and N‐termini, which vary in number and accessibility for different proteins. This work investigates the accuracy of estimation of protein concentration with SDS‐CGE‐LIF in real biological samples, where a different protein must be used as a standard. Sixteen purified proteins varying in molecular weight, structure, and sequence were labeled with FQ at constant mass concentration applying a commonly used procedure for SDS‐CGE‐LIF. The fluorescence of these proteins was measured using a spectrofluorometer and found to vary with a RSD of 36%. This compares favorably with other less sensitive methods for estimation of protein concentration such as SDS‐CGE‐UV and SDS‐PAGE‐Coomassie and is vastly superior to the equivalently sensitive silver stain. Investigation into the number of labels bound with UHPLC‐ESI‐QTOF‐MS revealed large variations in the labeling efficiency (percentage of labels to the number of labeling sites given by the sequence) for different proteins (from 3 to 30%). This explains the observation that fluorescence per mole of protein was not proportional to the number of lysines in the sequence.     

In‐tube solid‐phase microextraction capillary column packed with mesoporous TiO2 nanoparticles for phosphopeptide analysis

In this study, an in‐tube solid‐phase microextraction column packed with mesoporous TiO₂ nanoparticles, coupled with MALDI–TOF–MS, was applied to the selective enrichment and detection of phosphopeptides in complex biological samples. The mesoporous TiO₂ nanoparticles with high specific surface areas, prepared by a sol–gel and solvothermal method, were injected into the capillary using a slurry packing method with in situ polymerized monolithic segments as frits. Compared with the traditional solid‐phase extraction method, the TiO₂‐packed column with an effective length of 1 cm exhibited excellent selectivity (α‐casein/β‐casein/BSA molar ratio of 1:1:100) and sensitivity (10 fmol of a β‐casein enzymatic hydrolysis sample) for the enrichment of phosphopeptides. These performance characteristics make this system suitable for the detection of phosphorylated peptides in practical biosamples, such as nonfat milk.     

High-resolution 2-DE for resolving proteins, protein adducts and complexes in plasma

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.     

Equalizer technology followed by DIGE‐based proteomics for detection of cellular proteins in artificial peritoneal dialysis effluents

Peritoneal dialysis effluent (PDE) represents a rich pool of potential biomarkers for monitoring disease and therapy. Until now, proteomic studies have been hindered by the plasma‐like composition of the PDE. Beads covered with a peptide library are a promising approach to remove high abundant proteins and concentrate the sample in one step. In this study, a novel approach for proteomic biomarker identification in PDEs consisting of a depletion and concentration step followed by 2D gel based protein quantification was established. To prove this experimental concept a model system of artificial PDEs was established by spiking unused peritoneal dialysis (PD) fluids with cellular proteins reflecting control conditions or cell stress. Using this procedure, we were able to reduce the amount of high abundant plasma proteins and concentrate low abundant proteins while preserving changes in abundance of proteins with cellular origin. The alterations in abundance of the investigated marker for cell stress, the heat shock proteins, showed similar abundance profiles in the artificial PDE as in pure cell culture samples. Our results demonstrate the efficacy of this system in detecting subtle changes in cellular protein expression triggered by unphysiological stress stimuli typical in PD, which could serve as biomarkers. Further studies using patients’ PDE will be necessary to prove the concept in clinical PD and to assess whether this technique is also informative regarding enriching low abundant plasma derived protein biomarker in the PDE.     

The inter‐ and intra‐operator variability in manual spot segmentation and its effect on spot quantitation in two‐dimensional electrophoresis analysis

Separation of complex mixtures of proteins by 2‐DE is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for identifying alterations in protein expression within a given biological system. Software packages are designed for this purpose. The accurate definition of protein spot boundaries, using a suitable method of image segmentation, is a key requirement for image analysis. It is often necessary for operators to intervene manually to correct mistakes in spot segmentation; therefore operator subjectivity and differences in ability can weaken the analysis. We estimated the error in spot quantification after manual spot segmentation, which was performed by different operators, using two different software packages. Our results clearly show that this operation was associated with significant inter‐ and intra‐variability and an overestimation of subsequent spot intensity, especially when spots were weak. For comparative studies, we suggest separately analysing spots which have been manually segmented by imposing a requirement for at least a threefold difference in spot intensity in addition to use of statistical tests.