Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing

A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.     

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two-dimensional (2-D) liquid separations/mass mapping technique. This method uses a 2-D liquid separation of proteins from whole cell lysates coupled on-line to an electrospray ionization-time of flight (ESI-TOF) mass spectrometer to map the accurate intact molecular weight (M(r)) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed-phase high-performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI-TOF-MS provides an image of pI versus M(r) analogous to 2-D gel electrophoresis. Each protein is then identified based upon matrix-assisted laser desorption/ionization (MALDI)-TOF-MS peptide mapping and intact M(r) so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact M(r) together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 M(r) bands are detected over 17 pI fractions from pH 4 to 12 and a M(r) range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein M(r) is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein M(r) map may serve as a highly reproducible standard Web-based method for comparing proteins from related human cell lines.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

Alkylation power of free Immobiline chemicals towards proteins in isoelectric focusing and two-dimensional maps, as explored by matrix assisted laser desorption/ionization-time of flight-mass spectrometry

A number of Immobilines, with pK 1.0-10.3, were incubated with two proteins, bovine alpha-lactalbumin (pI 4.80) and chicken egg lysozyme (pI 9.32), at pH approximately 9-10 and the resulting solutions were examined by matrix assisted laser desorption/ionization mass spectrometry. The reflectron mode of the same technique was also used to analyze a number of tryptic digests of some solutions. The extent and the number of detected alkylation sites associated with the acidic protein were found to be linearly proportional to the pK values of the investigated Immobilines, an effect which was less evident for the basic protein. The high resolution measurements of some tryptic digests indicate the cysteine residues as the likely sites of alkylation. The implications of the present data for isoelectric focusing separations on immobilized pH gradients and for two-dimensional maps are discussed.     

Solid-liquid interfacial energy as a tool to estimate shifts in isoelectric points of adsorbed proteins on solid surfaces

This work reports the estimation of isoelectric points (pIs) of adsorbed amino acids and proteins on solid surfaces in the pH range between 3.5-11.0 from a measurement of solid/liquid interfacial energy. The values thus obtained are compared with the pIs determined in solution phase by other methods. Both glass and Teflon have been chosen as model solid surfaces. Close agreement between the reference pI values, obtained by the capillary isoelectric focusing and those obtained at solid/liquid interface is observed within an average difference of 0.04-0.08 pH unit when the pIs are above the pI of glass. For systems whose pIs are far away from that of glass (either in the acidic or highly alkaline range), a large shift in the isoelectric point is observed. In case of Teflon the pIs are closer to the reported values than at glass/liquid interface. This could be due to the fact that Teflon being a hydrophobic surface, its surface is dominated by dispersive forces, which may not be seriously affected by pH changes. The shift in the values at solid/liquid interface compared to that in solution have been examined using an 'image charge approach.'     

Parallel processing in the isoelectric focusing chip

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.     

Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides

In this work, we demonstrate the potential use of immobilized pH gradient isoelectric focusing as a first dimension in shotgun proteomics. The high resolving power and resulting reduction in matrix ionization effects due to analyzing peptides with almost the exact same physiochemical properties, represents a significant improvement in performance over traditional strong cation-exchange first-dimensional analysis associated with the shotgun proteomics approach. For example, using this technology, we were able to identify more than 6000 peptides and > 1200 proteins from the cytosolic fraction of Escherichia coli from approximately 10 microg of material analyzed in the second-dimensional liquid chromatography-tandem mass spectrometry experiment. Sample loads on the order of 1 mg can be resolved to 0.25 isoelectric point (pI) units, which make it possible to analyze organisms with significantly larger genomes/proteomes. Accurate pI prediction can then be employed using currently available algorithms to very effectively filter data for peptide/protein identification, and thus lowering the false-positive rate for cross-correlation-based peptide identification algorithms. By simplifying the protein mixture problem to tryptic peptides, the effect of specific amino acids on pI prediction can be evaluated as a function of their position in the peptide chain.     

Charge regulation in protein ion-exchange chromatography: development and experimental evaluation of a theory based on hydrogen ion Donnan equilibrium

An extension of the stoichiometric displacement (SD) model for the ion-exchange adsorption of dilute proteins is developed which accounts for the effects of hydrogen ion Donnan equilibrium on the protein charge. The ability of the new model to fit retention data when the fluid phase pH is near the protein pI and the effects of hydrogen ion Donnan equilibrium are important is examined using four different proteins and four different column packings. The results indicate that the model is able to fit retention data using values for the protein pI and the change in protein charge with pH at the pI, i.e., (dz/dpH)pI, that are significantly closer to the values of these parameters determined by isoelectric focusing and acid-base titrametry in free solution, respectively, as compared to the values obtained by determining the characteristic binding change as a function of pH using the traditional stoichiometric displacement model. This suggests that when the fluid phase pH is near the protein pI, charge regulation is an important cause of the discrepancy between the electrical charge of a protein in free solution and the characteristic binding charge from the stoichiometric displacement model. The results also indicate that for the case where the fluid phase pH is near the protein pI, the new model accounts for the effect of charge regulation during protein ion-exchange adsorption more accurately than previous models in the literature.     

Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax glass chips with length 70 mm, 300 microm width and 100 microm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22-5 microg microL(-1). Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.     

Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection

A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300?nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.     

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3)

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples.     

K-Means聚类分析法筛选柠檬香茅茎叶差异蛋白及鉴定

柠檬香茅含有大量的香茅精油,运用十分广泛,然而其茎、叶的精油含量却相差悬殊。 为探索柠檬香茅精油代谢相关的蛋白途径,本文对柠檬香茅旗叶、成熟叶及茎秆等材料进行精油含量、总蛋白含量测定及双向凝胶电泳(2-DE)表达谱分析,运用k-means聚类分析方法对2-DE电泳中差异蛋白斑点的丰度、等电点和相对分子质量进行聚类分析和讨论,结果表明,旗叶和茎秆上调表达的蛋白质斑点的聚类对于相对分子质量变化敏感,成熟叶上调表达蛋白质斑点对于丰度的变化较为敏感。 预测了精油代谢功能相关的蛋白质斑点15个,挖取预测蛋白质斑点通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)成功鉴定了9个蛋白质。 本研究为柠檬香茅精油的蛋白代谢途径提供新的基础信息及研究思路。     

Magnetic iron oxide nanoparticle enrichment of phosphopeptides on a radiate microstructure MALDI chip

Several methods can be used to improve the enrichment of phosphorylated proteins. In this paper, phosphopeptides were enriched using magnetic iron(II,III) oxide (magnetite, Fe(3)O(4)) nanoparticles (NPs) on a radiate microstructure silicon chip and then analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without further purification processes. We have developed a radiate microstructure chip on which samples can be concentrated for analysis by MALDI-TOFMS. The phosphoprotein digests and magnetic iron oxide NPs aqueous solution were deposited onto the central zone of the radiate microstructure silicon chip and enabled the on-chip enrichment of phosphopeptides. Microscopic analysis confirmed that the applied samples were confined to the central zone. Sample spots focused on the chip were much smaller than those on an unmodified plate with the same total volume. Different additives were used and optimized processes were performed to minimize non-phosphopeptides interference. These data collectively demonstrate that our on-chip phosphopeptide enrichment protocol is a rapid and easy-to-use method for phosphoproteome analysis.     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Isoelectric point determination by imaged CIEF of commercially available SARS-CoV-2 proteins and the hACE2 receptor

In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor-binding domain (RBD) with His- and Fc-tag, the S1 subunit with His-tag, the S1/S2 subunits with His-tag and the human angiotensin-converting enzyme 2 (hACE2) with His-tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS-CoV-2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.