Strong Anion Exchange Chromatography Research Articles

Strong anion‐exchange liquid chromatography coupled with isotope ratio mass spectrometry using a Liquiface interface

The introduction of liquid chromatography coupled with isotope ratio mass spectrometry (LC/IRMS) as an analytical tool for the measurement of isotope ratios in non-volatile analytes has somewhat simplified the analytical cycle from sample collection to analysis mainly due to the avoidance of the extensive sample processing and derivatisation that were necessary for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Here we test the performance of coupling strong anion exchange to IRMS using only the second commercially available interface; the Liquiface. The system was modified from installation specification to improve peak resolution in the interface and maintain peak separation from the column to the mass spectrometer. The system performance was assessed by the determination of sensitivity, accuracy and precision attained from carbohydrate separations. The system performed satisfactorily after modifications, resulting in maintenance of peak resolution from column to mass spectrometer. The sensitivity achieved suggested that approximately 150 ng carbon could be analysed with acceptable precision (<0.3 per thousand). Accuracy was maintained in the interface as determined by correlation with offline techniques, resulting in regression coefficient of r(2) = 0.98 and a slope of 0.99. The average precision achieved for the separation of seven monosaccharides was 0.36 per thousand. The integration of a carbonate removal device limited the effect of background carbon perturbations in the mass spectrometer associated with eluent gradients, and the coupling of strong anion-exchange chromatography with IRMS was successfully achieved using the Liquiface.

Proteome, Phosphoproteome, and Hydroxyproteome of Liver Mitochondria in Diabetic Rats at Early Pathogenic Stages

It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they were rendered diabetic. Altogether, we identified 1091 mitochondrial proteins, 228 phosphoproteins, and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in beta-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation, and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confronted T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of oxidative phosphorylation was immediately related to the overproduction of reactive oxygen species, which caused oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles such that mitochondrial proteins were more heavily hydroxylated during T2D development. Moreover, we observed a distinct depression of antiapoptosis and antioxidative stress proteins that might reflect a higher apoptotic index under the diabetic stage. We suggest that such changes in systematic metabolism were causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomics or genomics approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D.

Application of serum protein profiling in diagnosis, prognosis and evaluation of curative effect of pancreatic adenocarcinoma

To establish decision tree and logistic regression classification models for diagnosing pancreatic adenocarcinoma (PaCa) and for screening serum biomarkers related to evaluation of different stages and curative effects. Serum samples obtained from subjects with pancreatic adenocarcinoma (n = 58) and normal pancreas (n = 51) were applied to strong anion exchange chromatography (SAX2) chips for protein profiling by SELDI-TOF-MS to screen multiple serum biomarkers. Biomarker Wizard software and several statistical methods including algorithm of decision tree, logistic regression and ROC curves were used to construct the decision tree or logistic regression classification models. Average of 61 mass peaks were detected at the molecular range of 2000-30,000, ten decision trees with the highest cross validation rate were chosen to construct the classification models, which can differentiate PaCa from normal pancreas with a sensitivity of 83.3% and a specificity of 100%. Logistic regression was used to achieve the AUC (0.976 +/- 0.011, P < 0.001) with a sensitivity of 77.6% - 91.4% and a specificity of 92.2% - 100%. Six mass peaks were combined by logistic regression to achieve the AUC 0.897 +/- 0.054, 0.978 +/- 0.021 and 0.792 +/- 0.107 (P < 0.05) in the three groups (patients at stage I and II, stage II and III, stage III and IV). One mass peak (M/Z 4,016) was screened (P < 0.05) significantly between the preoperative and postoperative PaCa samples and the intensity decreased weeks after operation. Decision tree and logistic regression classification models of the mass peaks screened by SELDI-TOF-MS serum profiling can be used to differentiate pancreatic adenocarcinoma from normal pancreas, and is superior to CA 199. The detected mass peaks are helpful for the evaluation of curative effect and prognosis of pancreatic adenocarcinoma.

The phosphoproteome of Fusarium graminearum at the onset of nitrogen starvation

Fusarium graminearum grown under stress, such as nutrient deprivation, activates, among others, the trichothecene pathway that produces the mycotoxin deoxynivalenol and its derivatives. The kinase inhibitor staurosporine reduced the production of trichothecenes by 39% compared with control in vitro. On the other hand, phosphatase inhibitor okadaic acid increased the amount by 72% compared with the control in vitro. This suggests that phosphorylation events are involved in the signalling pathway, leading to the activation of the trichothecene pathway. Three approaches were used to study the phosphoproteome of F. graminearum under nitrogen-limiting conditions: 2-DE (2-DE: IEFxSDS-PAGE) in combination with MS protein identification; SDS-PAGE in combination with off-line IMAC and TiO(2) enrichment and gel electrophoresis LC-MS analysis; and a gel-free approach using strong anion exchange chromatography, IMAC and LC-MS. A total of 348 phosphorylation sites localized in 301 peptides from 241 proteins were identified. By 2-DE, 20 phosphoproteins were identified, nine of which underwent changes during the time course examined. Using gel electrophoresis LC-MS 231 phosphopeptides were identified from three samples (ten gel slices each) at time points of nitrogen starvation t=0, 6, and 12 h. The gel-free analysis added 70 peptides from 65 proteins to the total. Proteins of unknown function and enzymes of known function comprised the largest groups overall. Ten protein kinases and seven transcription factors were identified. This is the first reported phosphoproteome of F. graminearum.

Characterization of Heparin Impurities with HPLC-NMR Using Weak Anion Exchange Chromatography

A weak anion exchange (WAX) chromatography method coupled to online UV and NMR detection was developed for the separation and structural identification of heparin impurities. This work describes the separation of dermatan sulfate, chondroitin sulfate A, heparin, and the semisynthetic fully sulfated chondroitin sulfate, FSCS, through a displacement-based mechanism. The WAX separation requires significantly less salt than comparable strong anion exchange chromatography, facilitating NMR detection using a standard commercially produced flow probe. Retention and elution of both heparin and FSCS were found to be highly dependent on pH/pD and salt concentration. The elution time of each analyte could be controlled simply by adjusting a delay prior to introduction of the appropriate salt concentration, with little impact of the extent of the delay on chromatographic peak shape. Both on-flow and stop-flow proton ((1)H) NMR spectra were acquired to monitor and identify eluting components following separation. The approach described for the online separation of heparin and its potential impurities adds to the arsenal of techniques available for the rapid identification of new natural and/or intentional contaminants that may be introduced into the heparin supply chain.

Identification of serum biomarkers for pancreatic adenocarcinoma by proteomic analysis

Diagnosis of pancreatic adenocarcinoma (PaCa) at an early stage is important for successful treatment and improving the prognosis of patients. Serum samples were applied to strong anionic exchange chromatography (SAX) protein chips for protein profiling by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to distinguish PaCa from noncancer. The Wilcoxon rank-sum test, decision tree algorithm, and logistic regression were used to statistically analyze the multiple protein peaks. Sixty-one protein peaks between 2000 and 30,000 m/z ratios were detected to establish multiple decision classification trees for differentiating the known disease states. A sensitivity of 0.833 and a specificity of 1.000 were obtained in distinguishing PaCa from healthy controls and benign pancreatic diseases. Six protein biomarkers related to different PaCa TNM stages were detected (P < 0.01). One protein biomarker (m/z 4016) rich in PaCa had a down-regulated trend when preoperative and postoperative samples (P < 0.05) were compared. Three protein biomarkers (m/z 4155, 4791, and 28,068) were detected in the differential diagnosis of the three test groups (P < 0.05). A peak m/z 28 068 was identified as C14orf16 using ProteinChip immunoassay. C14orf166 levels were significantly higher in the serum of patients with PaCa compared with the control group using a sandwich immunoenzymatic system. Immunolabeling of tissue sections revealed that the C14orf166 protein was strongly expressed in tumor cells. The results suggest that SELDI-TOF-MS serum profiling is helpful for the diagnostic, prognostic or therapeutic effects of PaCa, which is superior to CA 19-9. The identified protein biomarker C14orf166 is a potential biomarker of PaCa.

Construction of protein profiling models for diagnosis of pancreatic carcinoma

Objective To establish diagnostic models for pancreatic carcinoma(PC)and to find out the biomarkers related to PC.Methods Serum samples obtained from subjects including PC patients,pancreatic benign disease patients and normal controls were examined with strong anionic exchange chromatography(SAX2)chips for protein profiling using surface enhanced laser desorption/ionization-time of flight-mass spectrometry(SELDI-TOF-MS).The decision tree models and logistic regression models for evaluating the value of serum biomarkers were assessed.SELDI immunoassay and ELISA were used to identify the biomarker and its level in serum respectively.Results Twentysix mass peaks were different between PC patients and normal controls(P＜0.0 1)and 16 mass peaks were different between patients with PC and with pancreatic benign disease(P＜0.05).The decision tree model had a sensitivity of 83.3%and a specificity of 100.0%in differentiation of PC,which was better than that of CA19-9 by ROC curve.There were significant differences in 6 mass peaks among different stages of PC(P＜0.01).Logistic regression model showed a sensitivity of 81.6%and a specificity of 80.6%in diagnosis of early PC.The M/Z 28068 protein was identified as C14orf166 with a sensitivity of more than 82%and a specificity of more than 88%in diagnosis of PC.Conclusions The diagnostic models based on SELDI-TOF-MS were superior to CA19-9 in diagnosis of PC.The identified biomarker C14orf166 is expected to play a role in the diagnosis of PC. Key words: Pancreatic neoplasms; Tumor markers; biological; Diagnosis; Spectrometry; mass; matrix-assisted laser desorption-ionization

Sequential Enrichment of Sulfated Glycans by Strong Anion-Exchange Chromatography Prior to Mass Spectrometric Measurements

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.

Deglutathionylation of 2-Cys Peroxiredoxin Is Specifically Catalyzed by Sulfiredoxin

Reversible protein glutathionylation plays a key role in cellular regulation and cell signaling and protects protein thiols from hyperoxidation. Sulfiredoxin (Srx), an enzyme that catalyzes the reduction of Cys-sulfinic acid derivatives of 2-Cys peroxiredoxins (2-Cys Prxs), has been shown to catalyze the deglutathionylation of actin. We show that deglutathionylation of 2-Cys Prx, a family of peroxidases, is specifically catalyzed by Srx. Using the ubiquitously expressed member of 2-Cys Prx, Prx I, we revealed the following. (i) Among its four Cys residues, Cys(52), Cys(83), and Cys(173) can be glutathionylated in vitro. Deglutathionylation with Cys mutants showed that Cys(83) and Cys(173) were preferentially catalyzed by Srx, with glutathionylated Srx as the reaction intermediate, whereas glutaredoxin I was more favorable for deglutathionylating Cys(52). (ii) Studies using site-directed mutagenesis coupled with binding and deglutathionylation activities revealed that Pro(174) and Pro(179) of Prx I and Tyr(92) of Srx are essential for both activities. Furthermore, relative to glutaredoxin I, Srx exhibited negligible deglutathionylation activity for glutathionylated cysteine and glutathionylated BSA. These results indicate that Srx is specific for deglutathionylating Prx I due to its favorable affinity for Prx I. To assess the biological relevance of these observations, we showed that Prx I is glutathionylated in A549 and HeLa cells under modest levels of H(2)O(2). In addition, the level of glutathionylated Prx I was substantially elevated in small interfering RNA-mediated Srx-knocked down cells, whereas the reverse was observed in Srx-overexpressing cells. However, glutathionylation of Prx V, not known to bind to Srx, was not affected by the change in Srx expression levels.

Arsenic speciation in cattail (Typha latifolia) using chromatography and mass spectrometry

Typha latifolia, commonly known as cattail, is widely used as traditional food and medicinal ingredients by indigenous people. There have been concerns over the high levels of total arsenic in cattail plants, but the chemical species of arsenic in cattail have not been characterized. We describe here the determination of arsenic species in the various compartments of cattail. Average concentrations of total arsenic from 9 to 19 cattail plants were 1120 microg/kg (range 68-2600 microg/kg) in the fine (hairy) roots, 575 microg/kg (range 16-1400) in the skin of tuber, 26 microg/kg (range 2-82) in the core of the tuber, 6 microg/kg (range 5-12) in the stem, and 420 microg/kg (range 4-1970) in the whole tuber. Speciation analysis using strong anion exchange, ion pairing, and strong cation exchange chromatography separation with MS detection revealed the presence of inorganic arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The two inorganic arsenic species accounted for >80% of the total arsenic. Further analyses of arsenic and iron concentrations showed a strong correlation between arsenic and iron in the fine roots and skin. These results suggest that arsenic and iron are colocalized (codeposited) in the skin of the cattail plants, consistent with the previous findings. The level of exposure to arsenic from the use of cattail as food and medicine can be substantially reduced by removing the skin of cattail.

Design of salt‐tolerant membrane adsorbers for viral clearance

Strong anion exchange chromatography has frequently been employed as a viral clearance step during downstream processing of biological therapeutics. When challenged with viruses having only slightly acidic isoelectric points, the performance of the anion exchange operation becomes highly dependent on the buffer salt concentration, with the virus log reduction value (LRV) dropping dramatically in buffers with 50-150 mM salt. In this work, a series of anion exchange membrane adsorbers utilizing alternative ligand chemistries instead of the traditional quaternary amine (Q) ligand have been developed that overcome this limitation. Four different ligands (agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide, and polyethyleneimine) achieved >5 LRV of bacteriophage PhiX174 (pI approximately 6.7) at pH 7.5 and up to 150 mM salt, compared to 0 LRV for the Q ligand. By evaluating structural derivatives of the successful ligands, three factors were identified that contributed to ligand salt tolerance: ligand net charge, ligand immobilization density on the membrane, and molecular structure of the ligand-binding group. Based on the results of this study, membrane adsorbers that incorporate alternative ligands provide a more robust and salt tolerant viral clearance-processing step compared to traditional strong anion exchange membrane adsorbers.

Evaluation of a solution isoelectric focusing protocol as an alternative to ion exchange chromatography for charge-based proteome prefractionation

Solution isoelectric focusing (sIEF) is evaluated relative to ion exchange chromatography (IEC) as a preferred charge-based prefractionation tool for proteome mixtures. While IEC is extensively employed for proteome prefractionation prior to MS analysis, we demonstrate here that conventional salt gradient IEC has significant shortcomings compared to sIEF. Here, we critically evaluated a custom eight-channel sIEF device for intact protein separation, relative to strong cation exchange (SCX) and strong anion exchange (SAX) chromatography. The resolution, recovery, and uniformity of separation obtained with our sIEF device were comparable or superior to that of optimized IEC separations. Most importantly for intact proteins, sIEF separations strongly correlate with the proteins’ isoelectric point, which contrasts with IEC where no correlation was observed. To validate the sIEF platform for proteome analysis, prefractionation through sIEF resulted in the confident identification of a greater number of proteins from yeast (211) following LC–MS/MS, relative to those obtained through SAX (173) or SCX (148).

Silicon isotopic composition of boreal forest vegetation in Northern Sweden

Thorough characterization of the Si isotopic composition of the terrestrial biogenic pool could potentially allow Si isotope information to be used when assessing the relative contributions from biogenic and mineral sources to soil water, plants and surface waters. In the present study, the Si isotopic compositions of major biomass components in a boreal forest in Northern Sweden were investigated, along with the relative contributions from exogenous Si incorporated in the plant structure. This was achieved using chemical purification and high-resolution multi-collector inductively coupled mass spectrometry (MC-ICP-MS) for the precise and accurate determination of the Si isotopic composition of plants. The technique, which relies on multi-elemental analysis of plant ashes and sample-specific HF-dissolution followed by strong-anion exchange chromatography, allows efficient separation of Si from matrix and interfering elements, while recovering in excess of 99% Si. The long-term instrumental reproducibility, expressed as two standard deviations (2 σ), for the isotopic reference material NBS28 ( n = 12) was 0.06‰ for δ 29Si and 0.12‰ for δ 30Si. Results for the analyses of composite plant samples for the eight most prolific species in the boreal forest yielded a surprisingly homogenous Si isotopic composition, expressed as δ 29Si (± expanded combined uncertainty) and δ 30Si, ranging from (− 0.15 ± 0.11)‰ to (0.13 ± 0.06)‰ and (− 0.31 ± 0.08)‰ to (0.22 ± 0.13)‰, respectively. Isotopic and elemental analyses of local airborne particulate material suggest that the exogenous Si contribution varies between < 1% and > 70%, indicating that the potential surface contribution must be considered during Si uptake studies. The present study thus provides evidence that thorough appreciation of the forms of Si in plants is an absolute requirement when assessing the plant impact on the Si cycle via the difference in dissolution kinetics for phytoliths and lithogenic Si.

On‐Line multitasking analytical proteomics: How to separate, reduce, alkylate and digest whole proteins in an on‐Line multidimensional chromatography system coupled to MS

An on-Line multidimensional system has been developed, consisting of pH gradient strong anion exchange chromatography of native proteins in the first dimension with subsequent trapping and on-column reduction/alkylation on C4 trap columns and RP separation of the alkylated proteins in the second dimension followed by on-column tryptic digestion and electrospray MS detection. The system was evaluated using model proteins and a human urine sample. Compared to the commonly used in-solution alkylation method, the developed on-column method provides an equivalent efficiency. The recovery from the C4 trap columns of the alkylated proteins relative to the native state was from 94 to 102%. On-column tryptic digestion was satisfactory for many, but not for all proteins. The whole analytical procedure was performed on-Line with packed capillary columns for a total time of 320 min for the first ion exchange fraction, with additional 60 min for each subsequent fraction.

On‐line Method for Identification of Native Proteins using pH‐Gradient SAX Chromatography and Reversed Phase Chromatography‐Mass Spectrometry of Tryptic Peptides

A method for identification of proteins based on their pI values and peptide mass fingerprint (PMF) using an on‐line system has been developed. Proteins were separated by pH‐gradient strong anion exchange (SAX) chromatography and trapped on C4 reversed phase (RP) columns. Trapped proteins were on‐line transferred to an immobilized trypsin reactor, and the resulting peptides were subsequently on‐line transferred to an RP analytical column via a combination of RP and weak cation exchange (WCX) trap columns. The tryptic peptides were identified by electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). Three model proteins were used for method development and basic human plasma proteins were subjected to identification. On‐column trypsination is compatible with high acetonitrile concentration. The analysis time was 180 min for the first fraction and an additional 80 min for each subsequent fraction selected for protein identification.

Large‐scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.

Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering

This study illustrates the application of asymmetrical flow field–flow fractionation (AF4) and light scattering analysis during the development of a gene delivery vehicle based on virus-like particles (VLPs) derived from the human polyoma JC virus. The analytical system was created by connecting an AF4 apparatus to the following detectors: diode array, fluorescence, multiangle light scattering, dynamic light scattering, and refractometer. From a single analysis, the molar mass, root mean square and hydrodynamic radii, composition, and purity of the sample could be determined. The VLPs were purified from baculovirus-infected Sf158 insect cells overexpressing the recombinant VP1 protein using weak anion exchange chromatography. The VLPs were dissociated to VP1 pentamers, and the contaminating DNA and proteins were removed using strong anion exchange chromatography. The gene delivery vehicle was created by reassembling the VP1 pentamers in the presence of the desired DNA. The newly formed VLPs encapsulated the DNA and were shown to be capable of delivering the gene of interest to target cells where it was translated into protein. This paper describes the scalable process that was derived to produce the VLPs and demonstrates how the AF4-based analytical characterization was indispensable during the development process.

Development of a two‐step chromatography procedure that allows the purification of a high‐purity anti‐histone H1 monoclonal immunoglobulin M antibody with immunosuppressant activity

In organ transplantation, the development of a novel immunosuppressant free of the need for permanent administration and any serious side effects has eagerly been awaited. We have previously reported that an anti-histone H1 polyclonal antibody has immunosuppressant activity. Here we prepared an anti-histone H1 monoclonal antibody as an analytical tool to elucidate its mechanism of immunosuppression. The isotype of this monoclonal antibody was immunoglobulin M. A monoclonal antibody prepared for administration to organ transplantation model animals should not contain any allogenic proteins and should have high purity. Therefore, we conducted a two-step chromatography procedure, consisting of strong anion-exchange chromatography and gel filtration chromatography, to purify an anti-histone H1 monoclonal immunoglobulin M antibody from the serum-free culture supernatant of hybridomas. Consequently, we successfully purified the monoclonal antibody at 96%, a purification rate at which its administration to organ transplantation model animals is possible.

Highly Efficient Phosphopeptide Enrichment by Calcium Phosphate Precipitation Combined with Subsequent IMAC Enrichment

A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.

The Behaviour of Reduced, Alkylated and Native Proteins in a pH-Gradient LC System

Two dimensional (2D) liquid chromatography (LC) separations of proteins can be obtained faster and more automated than traditional 2D gel electrophoresis. Previously we have described a 2D LC method for separation of native proteins with separation according to pI by pH-gradient strong anion exchange (SAX) chromatography in the first dimension, and according to hydrophobicity by reversed phase chromatography in the second dimension. Since there are few literature reports on the combination of reduced/alkylated proteins and modern LC, a basic study of the chromatographic properties of a few reduced /alkylated proteins was undertaken with a pH-gradient SAX chromatographic system. Proteins where the disulfide groups were reduced, but not alkylated, were also included. The conditions that separated native proteins according to pI could not be used for neither reduced nor reduced/alkylated proteins. High concentrations of urea (4–8 M) were needed in the mobile phase in order to obtain good peak shapes. Addition of urea had an undesired impact on both the retention of the proteins and the pH gradient profile, with the effect that little correlation between reported pI values and elution pH was found. The conclusion was that proteins should be separated in the native state if good pI–pH correlations are important, and in the alkylated state with urea if other considerations are more important.