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Over the years, multiple biomarkers have been used to aid in disease screening, diagnosis, prognosis, and response to therapy. As of late, protein biomarkers are gaining strength in their role for early disease diagnosis and prognosis in part due to the advancements in identification and characterization of a distinct functional pool of proteins known as proteoforms. Proteoforms are defined as all of the different molecular forms of a protein derived from a single gene caused by genetic variations, alternative spliced RNA transcripts and post-translational modifications. Monitoring the structural changes of each proteoform of a particular protein is essential to elucidate the complex molecular mechanisms that guide the course of disease. Clinical proteomics therefore holds the potential to offer further insight into disease pathology, progression, and prevention. Nevertheless, more technologically advanced diagnostic methods are needed to improve the reliability and clinical applicability of proteomics in preventive medicine. In this manuscript, we review the use of immunoaffinity capillary electrophoresis (IACE) as an emerging powerful diagnostic tool to isolate, separate, detect and characterize proteoform biomarkers obtained from liquid biopsy. IACE is an affinity capture-separation technology capable of isolating, concentrating and analyzing a wide range of biomarkers present in biological fluids. Isolation and concentration of target analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (CE) coupled to one or more detectors. IACE has the potential to generate rapid results with significant accuracy, leading to reliability and reproducibility in diagnosing and monitoring disease. Additionally, IACE has the capability of monitoring the efficacy of therapeutic agents by quantifying companion and complementary protein biomarkers. With advancements in telemedicine and artificial intelligence, the implementation of proteoform biomarker detection and analysis may significantly improve our capacity to identify medical conditions early and intervene in ways that improve health outcomes for individuals and populations.

The caffeine content of foods and beverages varies considerably, interfering with our ability to obtain valid interpretations in many human studies with regard to the mechanism of action(s) of caffeine and/or its metabolites. The rate of metabolism of caffeine and other xanthine drugs also varies greatly from one individual to another. Therefore, it is extremely important to develop accurate, reliable analytical methods to quantify caffeine and its metabolites in simple and complex matrixes. A simple method is described for the separation and characterization of caffeine and its major metabolites employing capillary electrophoresis (CE) coupled to ultraviolet-absorption and mass spectrometry (MS) detection. After optimization of the electrophoresis separation conditions, a reliable separation of caffeine and 11 of its major metabolites was achieved in 50 mM ammonium carbonate buffer, pH 11.0. The volatile aqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by MS. The CE method achieved baseline resolution for all 12 compounds in less than 30 min. The CE-MS method is suitable for use as a routine procedure for the rapid separation and characterization of caffeine and its metabolites. The usefulness of this method was demonstrated by the extraction, separation, and identification of caffeine and its 11 metabolites from normal urine samples. The urine specimens were first acidified to obtain optimum binding efficiency to the sorbents of the off-line, solid-phase extraction procedure employed here, and an acidified eluent solvent was employed for the desorption step to maximize the recovery of the bound analytes.

Capillary electrochromatography (CEC) is an emerging high-resolution separation technique that combines features of both capillary electrophoresis (CE) and micro-high-performance liquid chromatography (HPLC). CEC is capable of resolving neutral and ionic species with very high separation efficiencies because of its high selectivity and versatile properties. The difficulties in preparing reliable CEC columns and using CEC with regular CE instruments for routine analysis has limited its application. Nevertheless, many recent improvements of the technique, optimization of method development, and the potential use in pharmaceutical analysis and bioanalysis has made CEC a technique that is rapidly gaining acceptance. In this report, we have developed a CEC method for the rapid separation of a contraceptive drug substance, norgestimate, and its related degradation compounds. Total analysis time is reduced by more than 50% when compared with the current in-house HPLC method. The relative standard deviations for both the retention time and peak areas are less than 2%. Effects of other experimental parameters on the separation characteristics of norgestimate using CEC, such as effects of solvent composition, solvent strength, and buffer concentration, are discussed.

Diethylenetriaminepentaacetic anhydride (DTPA) conjugated to IgG (DTPA-IgG) and labeled with 111 In is useful for detecting focal sites of infection and inflammation (R.H. Rubin, A.J. Fischman, R.J. Callahan, B. Khaw, F. Keech, M. Ahmad, R. Wilkinson and H.W. Strauss, 111 In-labeled nonspecific immunoglobulin scanning in the detection of focal infection, N. Engl. J. Med., 321 (1989) 935–940). MACROSCINT ® contains DTPA-IgG formulated as a lyophile from a citrate buffer containing maltose. Exposure of both reconstituted and lyophilized MACROSCINT ® to intense light resulted in degradation primarily via formation of precipitating aggregates. However, lyophilized and reconstituted MACROSCINT ® responded differently to thermal stress. Reconstituted MACROSCINT ® subjected to thermal stress (65°C) also degraded through formation of precipitating aggregates. In contrast, exposure of lyophilized MACROSCINT ® to thermal stress (65°C) resulted primarily in an increase in the molecular size of the MACROSCINT ® DTPA-IgG monomer. This increase in molecular size was a function of both the moisture content in the vial and the amount of time for which the sample was stressed, but was not a function of the conjugation with DTPA. Monosaccharide analysis of the samples demonstrated that this increase in molecular size corresponded to an increase in the amount of glucose covalently attached to the IgG. These data suggest that the increase in molecular size as a function of thermal stress is due to the covalent attachment of maltose, which is a glucose disaccharide present in the lyophile as an excipient, to the IgG. This degradation pathway was only observed in the lyophile.

Humanized anti-Tac monoclonal antibody has been characterized by both SDS-PAGE and isoelectric focusing using traditional polyacrylamide methods. In addition to these techniques, capillary electrophoresis has been adapted to isoelectric focusing in a liquid phase. The humanized antibody has a pl in the range of 8.2–9.0, a region in which precast polyacrylamide or agarose gels are not stable in the 7–10 pH range. Experiments have been conducted to generate gradients in the 3–10 pH range and these gradients have successfully separated differently charged subspecies of humanized anti-TAC. Because of the simplicity in generating a variety of gradients, the power of resolution in different modes, its high efficiency, and the minimum sample volume requirements, capillary electrophoresis may become the method of choice for the routine characterization of therapeutic monoclonal antibodies and their stability profiles.

A preparation of a highly purified monoclonal antibody was subjected to capillary electrophoretic determination. Through the use of multiple parallel capillaries it was possible to increase both the detector response and sample load in direct proportion to the number of capillaries. While the amount of antibody solution assessed by ultraviolet detection was in the range of tenths of μg ml −1 , values in the range of tenths of ng ml −1 were obtained using fluorescence detection. The results are discussed in terms of their practical values for biological samples, targeted for human consumption as pharmaceutical drugs.

Background: The specific forms of pro–B-type natriuretic peptide (proBNP) that occur in human blood are not yet clear. We demonstrated the presence of several proBNP forms in human plasma with a new affinity chromatography method that can be used in combination with nano–liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC–ESI–MS/MS). Methods: For affinity chromatography, we coupled Fab′ fragments of polyclonal sheep antibodies specific for N-terminal proBNP (NT-proBNP) epitope 1–21 to silica beads. We connected a column (10 mm × 0.8 mm inner diameter) packed with these beads to a trypsin reactor and used a preconcentrator in combination with a fritless nanospray column to perform MS analyses of proBNP forms in preextracted and non-preextracted samples of plasma from patients with severe heart failure (HF). We used Western blotting in deglycosylation experiments to confirm the shifts in proBNP and NT-proBNP masses. Results: Tandem MS experiments demonstrated the presence of both NT-proBNP and circulating proBNP in preextracted samples of plasma from patients with severe HF, and Western blotting analyses revealed 2 bands of approximately 23 kDa and 13 kDa that shifted after deglycosylation to positions that corresponded to the locations of recombinant proBNP and synthetic NT-proBNP. Conclusions: We obtained clear evidence for circulating proBNP in patients with severe HF and provided the first demonstration of O-glycosylation of NT-proBNP. The higher molecular masses for NT-proBNP and proBNP observed in the Western blotting analyses than those expected from calculations can be explained by O-glycosylation of these peptides in vivo.

Several CE methodologies have been described for the analysis of rHuEPO in concentrated solutions, but the inherently limited concentration sensitivity of CE precludes the detection of EPO at the levels found in biological fluids. In this work, we have investigated an on-line immunoaffinity solid-phase extraction capillary electrophoresis (IA-CE) methodology for the selective preconcentration of EPO in diluted solutions. The preliminary results obtained using a custom-made immunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutaraldehyde-glass beads show the potential of this novel approach. The summarized findings are discussed in detail as a starting point for our ongoing investigations.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.

Cocaine and two other local anesthetics were applied directly into the nucleus accumbens for 20 min by diffusion from a 4 mm microdialysis probe in freely moving rats. Cocaine (7.3 mM) increased the extracellular concentration of dopamine (DA). Equimolar procaine did also, but was not as potent as cocaine. Equimolar lidocaine had no effect. The concentration of these drugs outside the probe as measured by capillary electrophoresis in vitro was about 28% of that inside the probe, i.e. 72% remained inside. However, an in vivo test showed that about 53% cocaine and procaine, and 37% lidocaine remained in the perfusion fluid after passing through a probe inserted in the brain. This suggests that in vivo about 68 nmol cocaine diffused into the nucleus accumbens (NAC) during the 20 min. Five conclusions are drawn: (1) this confirms our earlier finding that local injection of cocaine increases extracellular DA, but in this case the cocaine was infused via the probe without disturbing the animal; (2) the action of cocaine on dopamine terminals in the accumbens is independent of local anesthesia; (3) procaine may enhance mood by a cocaine-like effect; (4) capillary electrophoresis has potential for measuring cocaine levels in small samples and (5) in vitro calibrations are of limited value to evaluate in vivo performance of microdialysis probes.

Escherichia coli were activated by incubation with 1% glutaraldehyde, and the activated bacteria were then conjugated by incubation with IgG. About 66,000 molecules of IgG were bound per cell. The Escherichia coli -antibody conjugates were then used in competitive radioimmune assays. When a specific IgG fraction was used to prepare the conjugate, each cell bound about 16,000 molecules of antigen. With an antigen which had a specific activity of about 300 cpm/ng (or about 4.8 × 10 4 cpm/pmol), the assay was linear over a range of 40–300 ng/ml (0.3–2.0 pmol/ml). About 100 samples were assayed per day.

Prolyl 4-hydroxylase, the enzyme which synthesizes the 4-hydroxyproline found in collagens, was purified from human placenta. For this purpose, a new purification procedure was developed. The procedure involves chromatography on two DEAE-cellulose columns in addition to the previously developed affinity column. The new procedure makes it possible to obtain homogenous enzyme from tissues which contain low concentrations of prolyl 4-hydroxylase. When applied to human placenta, the purification was about 11,000-fold. An incidental observation was that when the original affinity column procedure was applied to placenta, the eluted enzyme peak contained large amounts of immunoglobulin, probably immunoglobulin G. On the basis of this observation, a simple procedure was developed for purifying immunoglobulin from human placenta or plasma.

A disulfide-bonded peptide with a molecular weight of about 100 000 was isolated from the medium of cultured chick embryo tendons. It was shown to be a trimer with two types of subunits in a 2:1 ratio, and tryptic fingerprinting and immunological evidence indicated that it was derived from the carboxy-terminal-precursor-specific region of procollagen. Amino acid analysis after reduction and alkylation indicated that the trimer contains about 30 residues of half-cystine involved in intrachain as well as interchain disulfide bonding. The interchain bonds could be reduced and alkylated under nondenaturing conditions. Carbohydrate analysis showed that each of the three peptide chains in the trimer contains about two residues of N-acetylglucosamine and about ten residues of mannose. This suggests the presence of one or two oligosaccharide units per chain.

Addition of ionic and nonionic detergents to whole homogenates of liver, kidney and lung prepared by a mild homogenization technique resulted in a two- to three-fold increase of prolyl hydroxylase activity. After subcellular fractionation of whole homogenates of liver, particulate and supernatant fractions were incubated in the presence and absence of triton X-100 and assayed for prolyl hydroxylase activity. All particulate fractions tested were able to release significant amounts of prolyl hydroxylase activity in the presence of triton. The release of enzyme activity by triton was observed with the 1000 × g and 17,000 × g supernatants but not with the 105,000 × g supernatant; thus indicating that detergent does not activate soluble enzyme nor make the substrate more accessible to hydroxylation by the enzyme during incubation. Rigorous homogenization of the 17,000 × g particulate fraction with the Polytron ST system resulted in a substantial loss of the amount of prolyl hydroxylase activity released by treatment with triton. These data suggest that a significant amount of prolyl hydroxylase activity is associated with membranes under physiological conditions.

Matrix-free cells obtained from chick embryo cartilage were incubated in the presence of α,α′-dipyridyl and radioactive mannose in order to examine the incorporation of mannose into the propeptide extensions of Type II procollagen. Cell proteins were digested with bacterial collagenase and the digests were examined by polyacrylamide gel electrophoresis. Radioactive mannose was found in fragments from both the N- and C-propeptides, and therefore the results provided the first indication that both these propeptides of Type II procollagen contain mannose. The results also supported previous indications that addition of carbohydrate to the propeptides of procollagen does not require folding of the collagen domain into a triple helix.