Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.     

Two-dimensional electrophoresis of small-molecular-weight proteins

We have developed a procedure for two-dimensional separation of small-molecular-weight (9000–30,000), acidic (pI 4–6) proteins that allows the use of strips cut from horizontal isoelectric-focusing slab gels for the first dimension, and discontinuous gels containing sodium dodecyl sulfate and high concentrations of urea in the second dimension. This technique facilitates the screening of large numbers of samples and the evaluation of electrofocusing artifacts. We emphasize measures to prevent major problems encountered in the use of this technique, particularly those caused by diffusion and aggregation. We also describe an extension of the method which allows the two-dimensional comparison of many samples in a selected narrow pH zone of interest.     

Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes.     

Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents.

It was shown that in linear polyacrylamide gradient gels migration distance of a given protein increases as a function of the square root of the time of electrophoresis. The linearity between these two parameters is demonstrated by the statistical analysis of experimental data obtained with proteins of different shapes and a wide range of electrophoretic mobility. The slopes of the regression lines calculated by this method can be utilized to determine the molecular weight of a nondenatured protein. In fact, there is a linear relationship between the log of the molecular weights and the log of the slopes for proteins with M_rs between 20,000 and 950,000.     

Two-dimensional polyacrylamide gel electrophoresis of membrane proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is one of the most powerful separation techniques for complex protein solutions. The proteins are first separated according to their isoelectric point, driven by an electric field across a pH gradient. The pH gradient necessary for the separation according to isoelectric point (pL) is usually established by electrophoresing carrier ampholytes prior to and/or concomitantly with the sample. The second dimension is usually a separation according to molecular size. Mostly this separation is performed after complete denaturation of the proteins by sodium dodecyl sulfate and 2-mercaptoethanol (SDS-PAGE). This standard method has considerable disadvantages when relatively hydrophobic membrane proteins are to be separated: cathodic drift, resulting in nonreproducible separation, and the denaturation of the protein, mostly making it impossible to detect native properties of the proteins after separation (e.g., enzymatic activity, antigenicity, intact multimers, and so on). The protocols presented here take care of most of these obstacles. However, there is probably no universal procedure that can guarantee success at first try for any mixture of membrane proteins; some experimentation will be necessary for optimization. Two procedures are each presented: a denaturing (with urea) and a nondenaturing method for IEF in immobilized pH gradient gels using Immobilines, and a denaturing (with SDS and 2-mercaptoethanol) and a nondenaturing technique (with CHAPS) for the second dimension. Essential tips and tricks are presented to keep frustrations of the newcomer at a low level.     

Two-dimensional agarose gel electrophoresis without gel manipulation

The apparatus and procedure to perform two-dimensional agarose gel electrophoresis without manipulating the gel used for the first electrophoresis (first-dimension gel) have been developed. The procedure is less complex, less damaging to first-dimension gels, and more precise than procedures that require manipulation of the first-dimension gel. When combined with gel-embedding techniques, the procedure presented can be used to perform the second electrophoresis in a gel different from the first-dimension gel. A first-dimension gel too dilute to be manipulated and a more concentrated gel for the second electrophoresis have been used to separate DNA open circles from a mixture of variable-length linear DNAs.     

Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

Two-dimensional electrophoresis of soybean root plasma membrane proteins solubilized by SDS and other detergents

Proteins solubilized from enriched soybean root plasma membrane with sodium dodecyl sulphate (SDS) and selected non-denaturing detergents (octyl-β-d-glucopyranoside, Zwittergent 312, Zwittergent 314, Zonyl FSK, and Nonidet P-40) were electrophoresed in two-dimensions by standard procedures. The basic electrophoretogram ‘fingerprint’ was similar for all detergents tested. However, differences in the total number of polypeptides resolved and the presence or absence of certain polypeptides on specific two-dimensional gels indicated some selectivity. Of all detergents tested, SDS solubilized the most polypeptides (ca 95) and provided the best resolution. The other detergents solubilized 50–80 polypeptides with varying resolution. Of those tested, octyl-β-d-glucopyranoside consistently provided the best balance between the number of polypeptides resolved (ca 70) and the level of resolution. The results suggest that selected detergents may prove useful in plant plasma membrane studies which require non-denaturing conditions.     

Two-dimensional gel electrophoresis of wool intermediate filament proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods have been used to provide high-resolution separation of wool intermediate filament proteins (IFPs). An improved method of extraction was developed based on a previously published method. The improved method for extraction eliminates the use of dialysis and freeze-drying between the extraction and rehydration steps, allowing the extraction and rehydration for the first dimension gel to be achieved in one day. Improvements to the method for maintaining reducing conditions and chaotrope constitution, combined with low %T polyacrylamide gels, allowed the high-resolution separation of the two keratin IFP families and their individual family members. The IFPs were separated to produce a clearly defined spot pattern of higher intensity, with numerous minor spots not previously observed, and a marked improvement in the vertical resolution. Further work to analyse the composition of each of the protein spots has been made much easier by being able to separate the IFPs into discrete spots.     

Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field.     

Detection of DNA-binding proteins in polyacrylamide gel after denaturing electrophoresis]

A simple method for detection of DNA-binding proteins is offered. These proteins can be revealed, following their electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gel containing labeled DNA, by washing the gel in buffer to remove SDS and to allow protein renaturation. Protein-free DNA is washed out, remaining in the DNA-binding proteins that restored their original characteristic. After autoradiography these proteins are seen as black bands (by one-dimensional gel electrophoresis) or spots (by two-dimensional gel electrophoresis) on a grey background. High sensitivity of the method is shown by using protein fractions of rat liver and a standard method.     

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability

The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability.     

Two-dimensional gel electrophoresis of 125I-labeled surface proteins of human fibroblasts.

Human fibroblasts were labeled by the 125I-lactoperoxidase technique and the sodium dodecyl sulfate soluble proteins examined by two-dimensional gel electrophoresis to determine the charge heterogeneity of the surface proteins and test for differences in surface proteins in several hereditary disorders. Approximately 80 polypeptides were observed. Those below 65 000 daltons tended to occur as single spots, while those of higher molecular weight were often present as a series of polypeptides of similar molecular weight (charge isomers). The possible role of these proteins in cell-cell recognition is discussed.     

Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Two-dimensional electrophoresis of plant proteins with phastsystem using nonequilibrium pH gradient separation.

We have adapted a two-dimensional electrophoretic technique described by P. Z. O'Farrell et al. (Cell 12, 1133-1142, 1977) to Phastsystem, resolving both acidic and basic proteins by using nonequilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. Protein separation was optimized for the analysis of plant proteins. The use of the Phastsystem apparatus reduced times of preparation and separation, allowing the rapid screening of plant proteins on a large scale of isoelectric points. This technique was used for the immunodetection and characterization of two stress-induced proteins in irradiated tomato leaves.     

Two-dimensional electrophoresis database of fluorescence-labeled proteins of colon cancer cells

We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.