Analysis of cross-linked human hemoglobin by conventional isoelectric focusing, immobilized pH gradients, capillary electrophoresis, and mass spectrometry.

Diaspirin cross-linked hemoglobin (DCLHb), a hemoglobin-based oxygen carrier exhibiting near physiological oxygen binding capability and devoid of nephrotoxic side effects, was previously found, by gel permeation, reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry, to consist of ca. 94% cross-linked product (reacted on the Lys 99 of two alpha-chains), accompanied by ca. 6% cross-linked Hb, which also reacted on the Lys 132 and/or Lys-144 of the beta-chains and a small amount of intermolecularly cross-linked dimers. However, conventional isoelectric focusing in carrier ampholyte buffers (CA-IEF) gave an unexpected spectrum of four major, almost equally represented, pI species in the pH range of 6.82-7.01, a band of mid-intensity with a pI of 7.11, and two minor components with pls of 6.73 and 6.77. This extraordinary polydispersity was reevaluated by other surface charge probes, such as immobilized pH gradients (IPG) and capillary zone electrophoresis (CZE) of native and denatured globin chains. IPGs of DCLHb gave the expected spectrum of bands, consisting of a main component (92%) with pl 7.337 and three additional minor bands, with lower pIs, representing ca. 8% of the total. These data were in agreement with CZE profiles of native DCLHb, which resolved, in addition to the main DCLHb peak, 3-4 minor components representing ca. 10% of the total. Also, CZE of denatured, heme-free globin chains gave the expected pattern with only traces of minor, extrareacted species. The latter technique, in addition to resolving alpha- and beta-globin chains in a 1:1 ratio in control Hb, resolved a free beta- and the alpha-alpha-dimer in DCLHb. In a 1:1 mixture of control and DCLHb, three peaks were observed, eluting in the order alpha-, alpha-alpha- and beta-globin chains. The identity of the major DCLHb and of the minor species was ascertained by mass spectrometry.     

Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants

Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification.     

Identification and characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry of a new variant hemoglobin, Mataro [beta134(H12) Val > Ala

This work illustrates the practical use of combined microbore reversed-phase high-performance liquid chromatography (RP-HPLC) with electrospray ionization mass spectrometry (ESI-MS) in protein identification. The approach consisted of the detection of the abnormal beta-globin chain by ESI-MS analysis of mixtures of intact globins, which simultaneously provided their molecular masses. Separation of the polypeptide globin chains was carried out using microbore C4 RP-HPLC on-line with ESI-MS. Direct peptide-mapping ESI-MS without previous chromatographic separation was performed in order to identify tryptic peptides from whole blood. For the sequence confirmation of the abnormal peptide containing the mutation point, C18 RP-HPLC tryptic separation of the globin mixture on-line with collision-induced dissociation (CID) fragmentation was done. The y series ions allowed the identification of the hemoglobin (Hb) variant as [beta134(H12) Val > Ala]. This new Hb was named Hb Mataró, after the city where it was detected.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

Capillary isoelectric focusing--reproducibility and protein adsorption

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3-15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long-time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self-amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)-coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused-silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.     

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech <Emphasis Type="Italic">Nephelopsis oscura</Emphasis>

The subunit structure of the giant, extracellular hexagonal bilayer (HBL) hemoglobin (Hb) from the leech Nephelopsis oscura was investigated by electrospray ionization mass spectrometry (ESI-MS) employing a maximum entropy deconvolution of its complex, multiply charged ESI spectra. The denatured unreduced Hb consisted of three monomer globin chains (M), a1 = 16535 Da, a2 = 17171 Da and a3 = 17315 Da, five nonglobin linker chains, L1 = 24512 Da, L2 = 24586 Da, L3 = 24979 Da, L4 = 25006 Da, and L5 = 25566 Da and two subunits of 32950 Da and 33125 Da. ESI-MS of the denatured, reduced Hb showed that the latter were disulfide-bonded heterodimers (D) of globin chains b1 = 16322 Da and b2 = 16499 Da with chain c = 16632 Da. Time-of-flight ESI-MS of the Hb at pH 3.8, 4.5, 5.0, 5.8 and 7.0 revealed a distribution of charge states from 32(+) to 37(+) with masses decreasing from 211 to 208.5 kDa with increase in cone voltage from 60 to 160 V, indicating the presence of a subassembly comprising 12 globin chains. The subunit composition 6M + 3D + 12h, where M = 16993 Da and D = 33004 Da are the weighted masses and h = 616.5 Da, provides a calculated mass, 208.37 kDa that is closest to 208.5 kDa. Our experimental findings are consistent with the bracelet model of HBL Hbs, verified by the recent low-resolution crystal structure of Lumbricus Hb, wherein an HBL arrangement of 12 globin dodecamer subassemblies is tethered to a central complex of 36 linker chains for a total mass of 208.37 x 12 + 24.94 x 36 = 3398 kDa.     

Mass spectrometric identification of a candidate biomarker peptide from the in vitro interaction of epichlorohydrin with red blood cells

The reaction products of epichlorohydrin with human alpha- and beta- globins, obtained through in vitro incubation of these compounds and red blood cells, were determined by using reversed-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization tandem mass spectrometry. The alpha-globin was much more reactive than the beta-globin. At low incubation ratios, approximating the order of magnitude of epichlorohydrin concentration as found in workplaces, the only modified peptide still detectable was the 62-90 belonging to the alpha-chain and carrying an incremental mass of 92 u on either His72 or His89. Given that the two peptides co-eluted in a single chromatographic peak during RP-HPLC separation, they could be chosen as suitable biomarkers for quantification in the setting up of a new methodology for the biological monitoring of persons occupationally exposed, replacing currently known procedures.     

Electrospray mass spectrometry in the clinical diagnosis of variant hemoglobins

A combination of mass spectrometric (MS) techniques [electrospray MS, liquid secondary ion MS (LSIMS) and MS-MS] has been used for variant hemoglobin (Hb) detection and characterization. Electrospray MS allowed analysis of mixtures of intact globins giving simultaneously the molecular weights (accuracy 1-2 Da) and information about relative amounts of globins present. Currently, 14 Da is the minimum molecular weight difference required experimentally to accurately measure different species present in a mixture of 15-16 kDa proteins. Thus 80 and 79% of the known variants of alpha and beta chains, respectively, can be detected in mixtures with their normal counterparts, including Hb S (molecular weight difference = 30 Da). Abnormal hemoglobins detected were fractionated by C4 reversed-phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested by trypsin. The tryptic peptides were separated by C18 reversed-phase HPLC and analyzed by LSIMS to narrow down the mutation site to a single peptide. The mass measured in LSIMS frequently corresponded to a unique structure, thus giving the unequivocal identification of the mutation and its site. Where there was ambiguity, tandem MS on a Kratos Concept four-sector instrument was used for sequencing the abnormal peptide. The practical use of the methodologies presented is illustrated through analysis and identification of Hb G-San Jose, Hb Stanleyville II, Hb S and Hb Willamette.     

Investigation of interaction between human hemoglobin A0 and platinum anticancer drugs by capillary isoelectric focusing with whole column imaging detection

CIEF with whole column imaging detection (WCID) was used to investigate the interaction of platinum-based anticancer drugs, cis-platinum(II) diamine dichloride (cisplatin) and [SP-4-2-{1R-trans)]-(1,2-cyclohexanediamine-N,N')[ethanedioata(2-)-O,O']platinum (oxaliplatin), with human hemoglobin A(0) (Hb). This technique facilitates the investigation and characterization of the formation of adducts between drugs and proteins. Cisplatin and oxaliplatin were mixed with the target protein at different concentrations (0:1, 1:1, 1:10, 1:50, and 1:100), and the reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48, and 72 h at 37 degrees C in a water-bath. The focused Hb-drug adduct profiles were imaged by WCID. At higher drug to protein molar ratios (for both oxaliplatin and cisplatin), the results exhibit significant changes in the peak shapes and heights, which may indicate the destabilization of the protein. However, the conformational change was less evident at lower molar ratios. In addition, a major pI shift was observed for the oxaliplatin reaction mixtures (for 1:10, 1:50, and 1:100 ratios). In comparison with previously reported findings obtained by other analytical methods, conclusions were drawn about the validity of CIEF as a simple and convenient method for the investigation of protein-drug interactions. These results may provide useful information for further understanding the activity and toxicity of these chemotherapeutic drugs and improving their clinical performance.     

毛细管等电聚焦方法及其应用

讨论了毛细管等电聚焦中所涉及的问题,如分离机理、电渗、迁移方法、检测器及其应用。由于毛细管等电聚焦操作方式的多样性,使其可适用于不同的仪器条件。非交联丙烯酰胺涂层能很好地消除电渗和蛋白吸附;而采用未处理的毛细管时,动态涂敷纤维素类亲水聚合物对碱性和中性蛋白亦能取得较好的分离效果。电荷耦合器件成像检测器尚待进一步发展才能成为常用的检测工具。对于复杂样品来说,仍需解决的问题是保证在较宽的ｐＨ范围内ｐＩ的线性。     

Review on screening and analysis techniques for hemoglobin variants and thalassemia

Thalassemia involves gene mutation that causes the production of an insufficient amount of normal structure globin chains while Hb variant involves gene mutation that causes the change in type or number of amino acid of the globin chain. It has been reported that some 200 million people worldwide had hemoglobinopathies of some sort. Attempts to develop effective and economical techniques for screening and analysis of thalassemia and Hb variants have become very important. In this review, we report the different techniques available, ranging from initial screening to extensive analysis, comparing advantages and disadvantages. Some indirect studies related to thalassemia indication and treatment follow-up are also included. We hope that information on these various techniques would be useful for some scientists who are working on development of a new technique or improving the existing ones.     

Globin chain separation by SDS polyacrylamide gel electrophoresis. Simple screening method for elongated hemoglobin chains

A simple method for the separation of hemoglobin chains from hemolysate or globin, by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is described. The alpha, beta, and gamma chains can be clearly separated from each other. The alpha chain has the highest mobility, the beta chain has a slower mobility than the gamma chain, while the delta chain has about the same mobility as the beta chain. Hemoglobins with elongated chains can easily be detected by this method. Tak-beta, elongated by 11 residues, moves much more slowly than betaA but is much faster than alpha Constant Spring which is elongated by 31 residues. Screening of several individuals with slow-moving hemoglobins using this method led to the finding of a case with Hb Tak-beta thalassemia and other carriers of Hb Tak.     

Quantitation of protein binding to the capillary wall in acidic, isoelectric buffers and means for minimizing the phenomenon

Notwithstanding the use of acidic, amphoteric, isoelectric buffers with isoelectric points (pI) in the pH 2-3 range, adsorption of proteins to the naked silica wall can be non-negligible. Two such buffers have been tested: iminodiacetic acid (IDA; pI 2.23, apparent pH 3.2 in 7 M urea) and aspartic acid (pI 2.77, apparent pH 3.7 in 7 M urea). Three potential quenchers of such interactions have been tested: hydroxyethylcellulose (HEC; number average molecular mass, Mr 27,000), TEPA (tetraethylenepentamine) and a novel, quatemarized piperazine [N(methyl-N-omega-iodobutyl)-N'-methylpiperazine] (Q-Pip), either alone or in binary and ternary mixtures. Human alpha- and beta-globin chains have been used as test proteins in capillary electrophoresis separations. It has been found that mixtures of these compounds are the worst possible remedy. E.g., a ternary mixture comprising 0.5% HEC, 0.5 mM TEPA and 1 mM Q-Pip still leaves behind 4.5% adsorbed protein onto the silica surface in runs in IDA buffer and 7 M urea (pH 3.2). Conversely, 0.5 mM TEPA or 1 mM Q-Pip, when used alone, minimize adsorption down to only 1.8% and 0.5%, respectively. When the same globin chain separations are performed in Asp and 7 M urea (pH 3.7), the situation is much worse: 44% protein is adsorbed in a ternary mixture of 0.5% HEC, 1 mM Q-Pip and 0.5 mM TEPA. However, when used alone, 0.5 mM TEPA and 1 mM Q-Pip reduce globin adsorption to levels of 8% and 5%, respectively. TEPA and Q-Pip are found to be in all cases the best quenchers of protein interaction to naked fused-silica; in addition they exhibit the unique property of smoothing the base-line and giving reproducible runs. The best method for desorbing bound protein was found to be an electrophoretic step consisting in driving sodium dodecylsulphate micelles from the cathodic reservoir.     

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.     

Dynamic capillary coatings with zwitterionic surfactants for capillary isoelectric focusing

The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C12N3SO3), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C16N3SO3) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein-ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C16N3SO3 and CAS U coatings were shown effective in CEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C16N3SO3 was effective in suppressing EOF at acidic and neutral pH conditions. C12N3SO3 however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R2 approximately equal to 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.     

Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li β83 (EF7)Gly → Cys by electrospray mass spectrometry

An electrospray mass spectrometric approach to the identification of a human hemoglobin (Hb) variant involving a Cys residue incorporation is presented. In Hb Ta-Li (beta83Gly --> Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor beta chain variant whose mass apparently was 1 Da less than the expected mass difference of 46 Da for a Gly --> Cys substitution. Reduction of the globin chains with dithiothreitol gave an intense monomer with the expected mass difference for the Gly --> Cys substitution. After reprocessing the original raw data from the denatured Hb and taking into account the possibility of dimer formation, a component was revealed whose mass was consistent with a disulfide linked dimer of Ta-Li beta globins. The mutation was localized to peptide betaT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly --> Cys substitution occurred at residue 83 of the beta chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.     

Analysis of protein mixtures by electrospray mass spectrometry: Effects of conformation and desolvation behavior on the signal intensities of hemoglobin subunits

The determination of solution-phase protein concentration ratios based on ESI-MS intensity ratios is not always straightforward. For example, equimolar mixtures of hemoglobin alpha- and beta-subunits consistently result in much higher peak intensities for the alpha-chain. The current work explores the origin of this effect. Under mildly acidic conditions (pH 3.4) alpha-globin is extensively unfolded, whereas beta-globin retains residual structure. Because of its greater nonpolar character, the more unfolded alpha-subunit can more effectively compete for charge. This leads to suppression of beta-globin signals under conditions where the protein ion yield is limited by the charge concentration on the initially formed ESI droplets. More balanced intensities are observed when operating under charge excess conditions and/or in a solvent environment where both proteins are unfolded to a similar degree (pH 2.2). However, even in these cases the overall alpha-globin peak intensity is still twice as high as that of the beta-subunit. The persistent imbalance under these conditions originates from the different declustering behaviors of the two proteins. A considerable fraction of beta-globin undergoes incomplete desolvation during ESI, thereby reducing the intensity of bare [beta + zH](z+) ions. When including the contributions of incompletely desolvated species, the overall alpha:beta ion intensity ratio is close to unity. The alpha:beta intensity imbalance can also be eliminated by a strongly elevated declustering potential in the ion sampling interface. In conclusion, important factors that have to be considered for the ESI-MS analysis of protein mixtures are (1) conformational effects, resulting in differential surface activities, and (2) dissimilarities in the protein desolvation behavior.     

Detection of the embryonic zeta chain in blood from newborn babies by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) using the large-pore Vydac C4 column has been used to detect and quantitate the embryonic zeta chain in blood samples of normal babies and of newborns with varying degrees of alpha chain deficiencies. The zeta chain eluted at the end of the chromatogram at about 130 min using a modified and extended gradient. Its identity was confirmed by structural analysis of zeta chain isolated from a blood sample of a fetus without active alpha globin genes, i.e. with hydrops fetalis (--/--). The quantity of zeta in normal babies is less than 0.7% [% of (alpha + zeta)] and is dependent upon the maturity of the baby as it was only present in babies with low levels of beta chain or hemoglobin (Hb) A. The presence of a zeta globin gene deletion [A. E. Felice et al., Hum. Genet., 73 (1986) 221; and P. Winichagoon et al., Nucleic Acids Res., 10 (1982) 5853] did not affect the level of zeta in the newborn. All babies with an alpha-thalassemia-2 heterozygosity, i.e. with three active alpha globin genes or -alpha/alpha alpha, had zeta in a range of 0.1-0.9%; again the level showed a negative correlation with that of the beta chain. Newborns with an alpha-thalassemia-2 homozygosity or -alpha/-alpha had a varying level of zeta of 0.3-2.3%, which did not correlate with the level of beta, suggesting that zeta chain production persists after birth in this condition. Macrochromatographic analyses in combination with RP-HPLC indicated that the zeta chain is present as zeta 2 gamma 2 or Hb Portland-I, as expected.     

Comprehensive two-dimensional separation system by coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.     

A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).