Detection and quantitation of recombinant granulocyte colony-stimulating factor charge isoforms: comparative analysis by cationic-exchange chromatography, isoelectric focusing gel electrophoresis, and peptide mapping.

Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln----Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.     

DIABLA: a new screening method for the discovery of protein targets

Dynamic isoelectric/anisotropy binding ligand assay (DIABLA) is a new method to identify proteins in a complex sample that bind to a molecule of interest. This is accomplished by first using capillary isoelectric focusing (cIEF) to separate the proteins in a capillary based on their isoelectric point. This separation is performed while the compound being tested is present in the separation buffer. When the proteins are focused, the entire capillary is scanned to identify regions of nonzero anisotropy, which are locations where the test compound is interacting with a focused protein band. DIABLA was demonstrated by observing the binding of fluorescein-tagged progesterone to an MCF-7 breast cancer cell lysate. The proteins were tagged with rhodamine to permit their observation and then focused in the presence of the tagged progesterone. Anisotropy measurements show that progesterone binds to six different proteins bands in the sample.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Automated sample introduction for an imaged capillary isoelectric focusing instrument via high-performance liquid chromatography sampling devices

Sample introduction of an imaged capillary isoelectric focusing (cIEF) instrument is fully automated by using commercially available high-performance liquid chromatography (HPLC) injection valves and autosamplers. Sample carryover can be controlled to under 1% when the valve and separation column are washed for 1 min between sample runs. The standard deviation of peak areas for 20 injections is 3.5%, which includes deviations created by the absorption imaging detector and the isoelectric focusing process inside the 75 μm I.D. column. Sample throughput is up to 10 samples per hour. The instrument has been applied to fast analysis of many proteins including monoclonal antibodies.     

Restriction isotyping of human apolipoprotein A-IV: rapid typing of known isoforms and detection of a new isoform that deletes a conserved repeat.

Genetic polymorphisms of apolipoprotein A-IV (apoA-IV) have been detected by isoelectric focusing of serum proteins. Because genetic variation in apoA-IV has significant effects on lipid risk factors, we used restriction enzyme isoform genotyping (restriction isotyping) to determine apoA-IV isoform genotypes at the DNA level for a large population (n = 509). In contrast to isoelectric focusing methods, restriction isotyping relies on nucleotide differences, enabling unambiguous typing of known isoforms and detection of new alleles that mimic other isoforms with shared charge properties. To determine genotypes for the common A-IV-1 isoform (Gln at aa position 360) and A-IV-2 isoform (360His), we used a mismatched primer for polymerase chain reaction (PCR) to introduce a restriction site (PvuII) that distinguishes each isoform. Using a portion of the same PCR reaction, we used HinfI to distinguish isoforms with Thr at position 347 (347Thr) versus Ser (347Ser). In surveys for these common genotypes, we detected heterozygotes for an allele with an insertion of 12 bp. Nucleotide sequencing showed that this allele is identical to the A-IV-0 isoform that inserts a hydrophilic repeat (Glu Gln Gln Gln) in a conserved region near the carboxy terminus. In addition, we discovered a new allele with a 12 bp deletion that removes a repeat (Glu Gln Gln Gln) from the same region. Nucleotide sequencing showed that this allele removes an acidic charge relative to A-IV-1, so we have named this isoform A-IV-2*. This isoform has not been discovered at the protein level, perhaps due to shared charge properties with A-IV-2 isoforms.     

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Clinical applications of capillary electrophoresis

Capillary electrophoresis (CE) is an extremely sensitive technique, which has been used in the clinical laboratory for almost 10 yr. The components of CE instrumentation are described, as are injection modes, buffers, and effects of electroosmotic flow. The modes of separation used in CE, namely, capillary zone electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic capillary chromatography, are explained. References for 26 different clinical applications of CE are included, among them assays that are used routinely as well as niche assays for specialized applications of CE. Verification of CE assays, current instrumentation, and future development of CE in the clinical laboratory are addressed.     

A First Tetraplex Assay for the Simultaneous Quantification of Total α-Synuclein,Tau, β-Amyloid42 and DJ-1 in Human Cerebrospinal Fluid

The quantification of four distinct proteins (α-synuclein, β-amyloid_1-42, DJ-1, and total tau) in cerebrospinal fluid (CSF) has been proposed as a laboratory-based platform for the diagnosis of Parkinson’s disease (PD) and Alzheimer’s disease (AD). While there is some clinical utility in measuring these markers individually, their usage in routine clinical testing remains challenging, in part due to substantial overlap of concentrations between healthy controls and diseased subjects. In contrast, measurement of different analytes in a single sample from individual patients in parallel appears to considerably improve the accuracy of AD or PD diagnosis. Here, we report the development and initial characterization of a first, electrochemiluminescence-based multiplex immunoassay for the simultaneous quantification of all four proteins (‘tetraplex’) in as little as 50 μl of CSF. In analytical performance experiments, we assessed its sensitivity, spike-recovery rate, parallelism and dilution linearity as well as the intra- and inter-assay variability. Using our in-house calibrators, we recorded a lower limit of detection for α-synuclein, β-amyloid₄₂, DJ-1, and t-tau of 1.95, 1.24, 5.63, and 4.05 pg/ml, respectively. The corresponding, linear concentration range covered >3 orders of magnitude. In diluted CSF samples (up to 1:4), spike-recovery rates ranged from a low of 55% for β-amyloid₄₂ to a high of 98% for DJ-1. Hillslopes ranged from 1.03 to 1.30, and inter-assay variability demonstrated very high reproducibility. Our newly established tetraplex assay represents a significant technical advance for fluid-based biomarker studies in neurodegenerative disorders allowing the simultaneous measurement of four pivotal makers in single CSF specimens. It provides exceptional sensitivity, accuracy and speed.     

Development of a Chip/Chip/SRM platform using digital chip isoelectric focusing and LC-Chip mass spectrometry for enrichment and quantitation of low abundance protein biomarkers in human plasma

Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ～1-2.5 ng/mL with a CV of ～13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.     

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.     

Isolation of a distinct Mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by CD1b-restricted T cells

Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.     

Capillary isoelectric focusing coupled with dynamic imaging detection: A one-dimensional separation for two-dimensional protein characterization

Here, we devised a novel approach for two-dimensional (2D) protein characterization using a single one-dimensional separation followed by a second characterization in the same instrument. The approach combines capillary isoelectric focusing (CIEF), which separates proteins according to pI, with dynamic imaging detection, which permits monitoring of protein diffusion in real time and thereby allows estimation of molecular weight from diffusion coefficient. Compared with classical 2D gel electrophoresis, the approach provided several significant advantages including speed and ease in operation and automation, while yielding comparable accuracy. The approach was applicable for protein samples of low to moderate complexity.     

Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and AD.     

Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA. The free PSA samples examined migrated as four to nine distinct, highly resolved peaks, indicating the presence of several isoforms differing in their oligosaccharide compositions. Overall, the use of CE provides a rapid, reproducible method for separation of PSA into its individual isoforms.     

Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing

Strategies reported for the separation of proteins in capillary zone electrophoresis and capillary isoelectric focusing are reviewed. The strategies are grouped into two categories: coated capillaries and buffer/sample additives. Success attained with each case and also, more importantly, the limitations of the methodology are discussed. Recent results from our own laboratory in the area of capillary isoelectric focusing in uncoated, fused silica capillaries using additives are summarized. The advantages and disadvantages of coated columns vs. additives are delineated.