First Dimension Separation Research Articles

Two-dimensional liquid chromatography separation of peptides using reversed-phase/weak cation-exchange mixed-mode column in first dimension

Selection of a first-dimensional separation method is very important in proteomic studies performed with two-dimensional liquid chromatography (2D-LC), since reversed-phase chromatography (RPLC) is usually chosen as the second-dimensional separation method in most studies. In this paper, we assess the potential use of mixed-mode reversed-phase/weak cation-exchange (RP/WCX) chromatography in 2D-LC proteomic studies. First, a new RP/WCX mixed-mode stationary phase (named C18WCX) was synthesized based on the polar-copolymerized approach, and a C18WCX column with separation efficiency comparative to conventional C18 columns was developed. This new mixed-mode column primarily provides hydrophobic interactions under acidic condition, but can offer hydrophobic and cation-exchange interactions under neutral and weak basic conditions. An off-line 2D-RP/WCX–RPLC system was established using the C18WCX column with a mobile phase of pH 6.5 in the first dimension and a C18 column with a mobile phase of pH 3.0 in the second dimension. The orthogonality of this two-dimensional system, evaluated through the separation of 123 tryptic peptides, was shown to be higher than that of the conventional 2D-RP–RPLC approach. Applying this 2D-LC method to rat brain samples, we identified 1031 proteins and 4397 unique peptides. In addition, this new two-dimensional method improved the identification of basic peptides. Therefore, we propose that this novel 2D-RP/WCX–RPLC system can be used as an alternative approach for the two-dimensional separation of peptides.

Evaluation of different two‐dimensional chromatographic techniques for proteomic analysis of mouse cardiac tissue

In proteomics experiments the first critical step after sampling is certainly sample preparation. Multidimensional chromatography techniques have emerged as a powerful tool for the large-scale analysis of such complex samples as biological samples. In order to evaluate these separation techniques, microgram quantities of protein extracted from mouse heart tissue were fractionated by four different chromatographic methods. Regarding peptide-level fractionation, the first dimension of separation was performed with high-pH reversed-phase chromatography (pH-RP) and strong cation exchange chromatography (SCX). Regarding protein-level fractionation, C(8) protein reversed-phase (C(8) -RP Prot) and high-recovery protein reversed-phase (hr-RP Prot) were used instead. The second dimension consisted of a reversed-phase nano-HPLC on-Chip coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The peptide-level pH-RP and the hr-RP Prot protein-level separations were the best methods, identifying 1338 and 1303 proteins, respectively. The peptide-level SCX, with 509 proteins identified, was the worst method.

Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO 2 pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Combinatorial use of offline SCX and online RP–RP liquid chromatography for iTRAQ-based quantitative proteomics applications

Extensive front-end separation is usually required for complex samples in bottom-up proteomics to alleviate the problem of peptide undersampling. Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based experiments have particularly higher demands, in terms of the number of duty cycles and the sensitivity, to confidently quantify protein abundance. Strong cation exchange (SCX)/reverse phase (RP) liquid chromatography (LC) is currently used routinely to separate iTRAQ-labeled peptides because of its ability to simultaneously clean up the iTRAQ reagents and byproducts and provide first-dimension separation; nevertheless, the low resolution of SCX means that peptides can be redundantly sampled across fractions, leading to loss of usable duty cycles. In this study, we explored the combinatorial application of offline SCX fractionation with online RP-RP applied to iTRAQ-labeled chloroplast proteins to evaluate the effect of three-dimensional LC separation on the overall performance of the quantitative proteomics experiment. We found that the higher resolution of RP-RP can be harnessed to complement SCX-RP and increase the quality of protein identification and quantification, without significantly impacting instrument time and reproducibility.

Novel Application of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) in Shotgun Proteomics: Comprehensive Profiling of Rat Kidney Proteome

In shotgun proteomics, multidimensional liquid chromatography (MDLC) is commonly used to reduce sample complexity and increase dynamic range of protein identification. Since reversed-phase chromatography is mostly used as the second-dimensional separation before mass spectrometric analysis, the improvement of MDLC primarily depends on the first dimension of separation. Here, we present a novel whole proteome analysis method that separates peptides based on ERLIC. Tryptic peptides were retained on a weak anion exchange column through ERLIC with a high organic mobile phase. They were then distributed into multiple fractions based on both pI and polarity through the simultaneous effect of electrostatic repulsion and hydrophilic interaction when eluted using a salt-free pH gradient of increasing water content. Applying this to rat kidney tissue, we identified 4821 proteins and 30 659 unique peptides with high confidence from two replicates using LTQ-FT. This was 36.2% and 64.3% higher, respectively, than was obtained with the widely used SCX separation mode. Notably, the identification of both highly hydrophobic and basic peptides increased over 120% using the ERLIC method. The results indicate that ERLIC is a promising alternative to SCX as the first dimension of MDLC. In total, 5499 proteins and 35 847 unique peptides of rat kidney tissue are characterized.

Comprehensive two-dimensional liquid chromatography: Ion chromatography × reversed-phase liquid chromatography for separation of low-molar-mass organic acids

In the work presented here a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Ion chromatography is chosen for the first-dimension separation and reversed-phase liquid chromatography is chosen for the second-dimension separation mode. The coupling of these modes is made possible by neutralising the first-dimension effluent, containing KOH, prior to transfer to the second-dimension reversed-phase column. A test mixture of 24 low-molar-mass organic acids is used for optimisation of the system. Three food and beverage samples were analysed in order to evaluate the developed methodology, the resulting two-dimensional separation is near-orthogonal, the set-up is simple and all instrumental components are available commercially. The method proved to be robust and suitable for the analysis of wine, orange juice and yogurt.

Workflow Comparison for Label-Free, Quantitative Secretome Proteomics for Cancer Biomarker Discovery: Method Evaluation, Differential Analysis, and Verification in Serum

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.

Isobaric Tagging-Based Selection and Quantitation of Cerebrospinal Fluid Tryptic Peptides with Reporter Calibration Curves

In the past few years, mass spectrometry (MS) has emerged as an efficient tool for the multiplexed peptide and protein concentration determination by isotope dilution. Despite the growing use of isobaric tagging to perform relative quantitation for the discovery of potential biomarkers in biological fluids, no real application has so far been presented for their absolute quantitation. Isobaric tandem mass tags (TMTs) were used herein for the selection and quantitation of tryptic peptides derived from brain damage related proteins in cerebrospinal fluid (CSF). Proteotypic tryptic peptide analogues were synthesized, prepared in four reference amounts, differentially labeled with four isobaric TMTs with reporter-ions at m/z = 128.1, 129.1, 130.1, and 131.1, and mixed with CSF sample previously labeled with TMT 126.1. Off-gel electrophoresis (OGE) was used as first-dimension separation of the pooled sample. The resulting fractions were analyzed with reversed-phase liquid chromatography (RP-LC) tandem mass spectrometry (MS/MS), using tandem time-of-flight (TOF/TOF) and hybrid linear ion trap-orbitrap (LTQ-OT) instruments. Under collision-induced dissociation (CID) or higher-energy C-trap dissociation (HCD), the release of the reporter fragments from the TMT-labeled peptide standards provided an internal calibration curve to assess the concentration of these peptides in the CSF. This tool also allowed identifying selectively these peptides in CSF as only the targeted peptides showed specific fragmentation pattern in the TMT reporter-ion zone of the tandem mass spectra. Assays for the concentration measurements of peptides from PARK7, GSTP1, NDKA, and S100B proteins in CSF were further characterized using this novel, efficient, and straightforward approach.

Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

BackgroundCreatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach.MethodsDifferentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH.ResultsThe identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH2 oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium.ConclusionsThe explorative approach of this study combined with the determination of a decreased intracellular DCFH2 oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress.

Analysis of potential genotoxic impurities in pharmaceuticals by two-dimensional gas chromatography with Deans switching and independent column temperature control using a low-thermal-mass oven module

The analysis of potential genotoxic impurities (PGIs) in active pharmaceutical ingredients (APIs) is a challenging task. The target limit of detection for a PGI in an API is typically 1 ppm (1 microg/g API). This is about 500 times lower than for classical impurity analysis. Consequently, analytical methods for trace analysis, mostly in combination with MS detection, need to be applied for the qualitative and quantitative determination of these impurities. A two-dimensional capillary GC method is presented that can be used for the determination of some target PGIs. A concentrated solution of the API sample is directly introduced in the GC-MS system, using an apolar column for first-dimension separation. The fraction (heart-cut) containing the PGIs is transferred to a second capillary column, installed in a low-thermal-mass oven (LTM). The LTM focuses the heart-cut(s) and allows independent temperature-programmed analysis with a polar second-dimension column. The API, solvent, and derivatization agents are not introduced in the second column or in the MS detector, avoiding contamination, column degradation, and target analyte peak detection/integration issues. The performance of this set-up is illustrated by the analysis of some Michael-reactive acceptor PGIs and haloalcohols in carbamazepine as test matrix. Excellent reproducibility (<10% RSD) at the low parts per million level and low detection limits (<1 ppm) were obtained.

Comparative analysis of OFFGel, strong cation exchange with pH gradient, and RP at high pH for first‐dimensional separation of peptides from a membrane‐enriched protein fraction

The analysis and quantitation of membrane proteins have proved challenging for proteomics. Although several approaches have been introduced to complement gel-based analysis of intact proteins, the literature is rather limited in comparing major emerging approaches. Peptide fractionation using IEF (OFFGel), strong cation exchange HPLC using a pH gradient (SCX-pG), and RP HPLC at high pH, have been shown to increase peptide and protein identification over classic MudPIT approaches. This article compares these three approaches for first-dimensional separation of peptides using a detergent phase (Triton X-114) enriched membrane fraction from mouse cortical brain tissue. Results indicate that RP at high pH (pH 10) was superior for the identification of more peptides and proteins in comparison to the OFFGel or the SCX-pG approaches. In addition, gene ontology analysis (GOMiner) revealed that RP at high pH (pH 10) successfully identified an increased number of proteins with "membrane" ontology, further confirming its suitability for membrane protein analysis, in comparison to SCX-pG and OFFGel techniques.

Detection issues in two-dimensional on-line chromatography

The dilution of analytes during their migration through two-dimensional liquid chromatographic systems was investigated. Simplified equations for the calculation of the dilution factors in the first and second dimension columns were derived considering the variance of the injected sample, the flow rates, the split ratio, the volume of the intermediate sample loop and the sampling time of the first-dimension effluent. The net dilution factor is the product of the dilution factors in the two single dimensions. It is between 200 and 300 in typical two-dimensional liquid chromatographic (2D-LC) separations. These values are less pessimistic than those reported previously [1]. It was shown that the fraction of organic modifier or stronger eluent component in the injected sample has a significant effect on the widths and heights of the eluted peaks. Analytes with high retention factors and high molecular weights are less sensitive toward this effect than those having small retention factors. These results suggest that the optimization of the experimental conditions of two dimension separations must be made from the points of view of both the retention factor and the detector response factor. The frequency of sampling of the eluent of the first-dimension separation does not have a significant effect on the detection limit of a 2D system if the split ratio is adjusted in the ratio of the frequency of sample collection.

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2-D-HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi-preparative HPLC (C18 column 250x10 mm, 5 microm) as a first dimension of separation to isolate phenolic fractions from commercial extra-virgin olive oils and CE coupled to TOF-MS (CE-TOF-MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF-MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2-D-HPLC-CE-MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra-virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti-carcinogenic capacity.

Generation and Limitations of Peak Capacity in Online Two-Dimensional Liquid Chromatography

The different operating conditions of an online two-dimensional liquid chromatographic separation (2D-LC), such as the length of the column, the linear velocity and the composition of the mobile phase used in the second dimension, its initial organic content if this separation is carried out in gradient elution, the number of fractions of the first column eluent collected, and the analysis time of the first dimension all affect the achievable separation power of 2D-LC online systems. The influences of these factors on the separation performance were investigated, and an equation was derived for the calculation of the achievable peak capacity in online 2D-LC assuming (1) that the option of undersampling the first-dimension separation is acceptable, (2) that the solutes follow linear-solvent-strength behavior, and (3) that all the separations are made in gradient elution. This theoretical discussion shows that (1) highly efficient separations made with online 2D-LC require the second-dimension peaks to be very narrow, (2) the separation power of 2D-LC systems is maximum for an optimum number of fractions collected in the first dimension, (3) higher peak capacities can be achieved by using shorter second-dimension columns and collecting a relatively large number of fractions, (4) the achievable 2D peak capacity is maximum for a certain eluent flow rate and column length of the second-dimension column, and (5) the maximum achievable peak capacity increases with decreasing velocity and initial organic content of the second-dimension eluent. As a consequence, due to the time restriction of the second-dimension gradient time, online 2D-LC schemes cannot realistically afford peak capacities exceeding 10,000, even if they are implemented with exceptionally efficient columns and if long analysis times are accepted.

Comparative proteomic study of two closely related ovarian endometrioid adenocarcinoma cell lines using cIEF fractionation and pathway analysis

The proteomic profiles from two distinct ovarian endometrioid tumor-derived cell lines, (MDAH-2774 and TOV-112D) each with different morphological characteristics and genetic mutations, have been studied. Characterization of the differential global protein expression between these two cell lines has important implications for the understanding of the pathogenesis of ovarian endometrioid carcinoma. In this comparative proteomic study, extensive fractionation of peptides generated from whole-cell trypsin digestion was achieved by coupling cIEF in the first-dimensional separation with capillary LC (RP-HPLC) in the second dimensional separation. Online analysis was performed using tandem mass spectra acquired by a linear ion trap mass spectrometer from triplicate runs. A total of 1749 and 1955 proteins with protein probability above 0.95 were identified from MDAH-2774 and TOV-112D after filtering through Peptide Prophet/Protein Prophet software. Differentially expressed proteins were further investigated by ingenuity pathway analysis (IPA) to reveal the association with important biological functions. Canonical pathway analysis using IPA demonstrates that important signaling pathways are highly associated with one of these two cell lines versus the other, such as the PI3K/AKT pathway, which is found to be significantly predominant in MDAH-2774 but not in TOV-112D. Also, protein network analysis using IPA highlights p53 as a central hub relating to other proteins from the connectivity map. These results illustrate the utility of high throughput proteomics methods using large-scale proteome profiling combined with bioinformatics tools to identify differential signaling pathways, thus contributing to the understanding of mechanisms of deregulation in neoplastic cells.

Repeatability of peptide identifications in shotgun proteome analysis employing off‐line two‐dimensional chromatographic separations and ion‐trap MS

The repeatability of peptide identifications in shotgun proteome analyses employing strong cation-exchange-xion-pair RP HPLC hyphenated to ESI MS/MS was compared to an alternative scheme, comprising high-pH RP chromatography combined with low-pH ion-pair RP chromatography. Equivalent results were obtained with both methods in proteome analysis of Corynebacterium glutamicum. From a total number of 1350 to 1850 peptides identified in triplicate analyses of five consecutive fractions chosen from the first-dimension separation, 41-45% of the peptides were identified three times, whereas 16-22 and 37-39% of the peptides were identified only twice or once, respectively. A comparison of the repeatability of peptide identifications from complex samples upon 1- or 2-D chromatographic separation revealed that an additional separation dimension decreases the repeatability by approximately 25%.

A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

Genetic polymorphism of an alpha 1-protease inhibitor in mink plasma.

Two-dimensional electrophoretic analysis of mink (Mustela vison Schreber) plasma proteins was done by a first-dimension separation in agarose gel (pH 8.6), followed by a second dimension in horizontal polyacrylamide gel (pH 9.0). Two α1-globulins, designated Pi-1 and Pi-2, and another protein, designated Pi-3 were found to inhibit the esterolytic activity of bovine trypsin and bovine chymotrypsin. The Pi-1 segregation analysis in limited family data supported the hypothesis that the three Pi-1 types observed were controlled by two codominant, autosomal alleles. Each of the Pi-1 homozygote types showed one strong, broad fraction while the Pi-1 heterozygote type had two fractions. The electrophoretic mobility and patterns of Pi-1 indicated that this protein was most probably identical to the polymorphic post albumin (Pa) in mink plasma, reported by Saison in 1968.

Two-dimensional gel electrophoresis of dog plasma proteins: genetic polymorphism of an alpha 1-protease inhibitor and another postalbumin.

Two-dimensional electrophoretic analysis of dog plasma proteins was done by a first-dimension separation in agarose gel (pH 5.4) followed by a second-dimension separation in horizontal poly-acrylamide gel (pH 9.0). Two groups of α1-globulins, designated Pi-1 and Pi-2, were found to be protease inhibitors. Extensive genetic polymorphism of Pi-1 and of another unidentified α-globulin, tentatively designated postalbumin (Pa), was observed. Family data supported the hypothesis that Pi-1 and Pa polymorphisms were controlled by codominant, autosomal alleles. Three Pi-1 alleles and two Pa alleles were observed in 60 breeds (1119 dogs) studied. Each of the Pi-1 alleles gave rise to a single strong fraction while each of the Pa alleles showed a major and a minor anodic fraction. The present study, along with earlier reports, showed that some of the proteins and enzymes (albumin, transferrin, eserine resistant esterase, α1-protease inhibitor and postalbumin) in plasma exhibit considerably more polymorphism than that reported for hemoglobin and some of the red cell and tissue enzymes of the domestic dog.