Proteins In Biological Matrices Research Articles

Western blotting: evolution of an old analytical method to a new quantitative tool for biomarker measurements.

Western blotting (WB) is a widely used laboratory technique for detecting specific proteins in biological matrices. Recent advances in antibody production, automation, gel and membrane manufacturingand highly sensitive detection platforms have transformed WB from a labor-intensive and qualitative method into a highly reproducible and quantitative assay suitable for biomarker detection. Despite these significant improvements in the capabilities and efficiency of WB, there remain challenges that hinder its widespread application as aresearch, diagnostic (in two-tiered assays like Lyme disease testing)and drug development tool. This article describesrecent innovations introduced to WB methodology and the remaining challenges that prevent its wider adoption for biomarker measurements throughout the drug development process.

Quantification of Proteins in Blood by Absorptive Microtiter Plate-Based Affinity Purification Coupled to Liquid Chromatography-Mass Spectrometry.

Liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly used for quantification of proteins in blood. This development is prompted by ongoing improvements in detection sensitivities of LC-MS instruments and corresponding sample preparation workflows. The combination of immunoaffinity enrichment and targeted LC-MS detection is a notable analytical platform in this regard as it allows for the quantification of low abundance proteins in biological matrices like plasma and serum. Here, we describe such hybrid methods which are based on the enrichment of proteins with antibodies or affimers coupled to adsorptive microtiter plates, the proteolytic digestion of enriched proteins to release protein-specific peptides, and the detection of these peptides by microflow LC coupled to selected reaction monitoring MS.

Exploiting His-Tags for Absolute Quantitation of Exogenous Recombinant Proteins in Biological Matrices: Ruthenium as a Protein Tracer.

Metal labeling and ICP MS detection offer an alternative to commonly accepted techniques that are currently used to quantitate exogenous proteins in vivo, but modifying the protein surface with metal-containing groups inevitably changes its biophysical properties and is likely to affect trafficking and biodistribution. The approach explored in this work takes advantage of the presence of hexa-histidine tags in many recombinant proteins, which have high affinity toward a range of metals. While many divalent metals bind to poly histidine sequences reversibly, oxidation of imidazole-bound CoII or RuII is known to result in a dramatic increase of the binding strength. In order to evaluate the feasibility of using imidazole-bound metal oxidation as a means of attaching permanent tags to polyhistidine segments, a synthetic peptide YPDFEDYWMKHHHHHH was used as a model. RuII can be oxidized under ambient (aerobic) conditions, allowing any oxidation damage to the peptide beyond the metal-binding site to be avoided. The resulting peptide-RuIII complex is very stable, with the single hexa-histidine segment capable of accommodating up to three metal ions. Localization of RuIII within the hexa-histidine segment of the peptide was confirmed by tandem mass spectrometry. The RuIII/peptide binding appears to be irreversible, with both low- and high-molecular weight biologically relevant scavengers failing to strip the metal from the peptide. Application of this protocol to labeling a recombinant form of an 80 kDa protein transferrin allowed RuIII to be selectively placed within the His-tag segment. The metal label remained stable in the presence of ubiquitous scavengers and did not interfere with the receptor binding, while allowing the protein to be readily detected in serum at sub-nM concentrations. The results of this work suggest that ruthenium lends itself as an ideal metal tag for selective labeling of His-tag containing recombinant proteins to enable their sensitive detection and quantitation with ICP MS.

Development of an automated, interference-free, 2D-LC-MS/MS assay for quantification of a therapeutic mAb in human sera.

Hybrid LC-MS/MS assays are increasingly used to quantitate proteins in biological matrices. These assays involve analyte enrichment at the protein level. Although suitability has been demonstrated, they are limited by the lack of appropriate affinity reagents and may suffer from interferences caused by binding proteins or antibodies. An online stable isotope standards and capture by anti-peptide antibodies assay was developed, which involves tryptic digestion of a therapeutic monoclonal antibody in human serum to destroy interfering proteins followed by enrichment using high affinity peptide antibodies. The assay was validated and compared with a standard ligand-binding assay currently used for quantification. The data show that the stable isotope standards and capture by anti-peptide antibodies-2D-LC-MS/MS assay can be used as an alternative method for measurement of monoclonal antibodies in clinical samples.

Reagent-free LC-MS/MS-based pharmacokinetic quantification of polyhistidine-tagged therapeutic proteins.

Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS. A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.

Techniques for quantitative LC-MS/MS analysis of protein therapeutics: advances in enzyme digestion and immunocapture.

LC-MS/MS has been investigated to quantify protein therapeutics in biological matrices. The protein therapeutics is digested by an enzyme to generate surrogate peptide(s) before LC-MS/MS analysis. One challenge is isolating protein therapeutics in the presence of large number of endogenous proteins in biological matrices. Immunocapture, in which a capture agent is used to preferentially bind the protein therapeutics over other proteins, is gaining traction. The protein therapeutics is eluted for digestion and LC-MS/MS analysis. One area of tremendous potential for immunocapture-LC-MS/MS is to obtain quantitative data where ligand-binding assay alone is not sufficient, for example, quantitation of antidrug antibody complexes. Herein, we present an overview of recent advance in enzyme digestion and immunocapture applicable to protein quantitation.

Rapid isolation and detection of erythropoietin in blood plasma by magnetic core gold nanoparticles and portable Raman spectroscopy

Isolating, purifying, and identifying proteins in complex biological matrices are often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesized, characterized, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15min. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles’ surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1min sample measurement time. From the Clinical EditorThe rapid detection of recombinant human erythropoietin (rHuEPO) is important in competitive sports to screen for doping offences. In this article, the authors reported their technique of direct surface enhanced Raman spectroscopy (SERS) detection using magnetic core gold nanoparticles functionalized with recombinant human erythropoietin-specific antibody. The findings should open a new way for future detection of other proteins.

Surfing Transcriptomic Landscapes. A Step beyond the Annotation of Chromosome 16 Proteome

The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.

Pellet Digestion: A Simple And Efficient Sample Preparation Technique For Lc–MS/MS Quantification of Large Therapeutic Proteins in Plasma

There is a need for a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma. The sample preparation technique presented here is based upon trypsin digestion of the pellet obtained following precipitation of the protein analyte from plasma. The pellet digestion technique was shown to facilitate efficient digestion of large therapeutic proteins, with concomitant removal of a substantial amount of potentially problematic plasma phospholipids. The technique was successfully applied to a pharmacokinetic study of a large therapeutic protein. This simple sample preparation approach will be beneficial to bioanalytical laboratories engaged in the LC-MS/MS quantification of large therapeutic proteins in biological matrices.

Direct detection of native proteins in biological matrices using extractive electrospray ionization mass spectrometry

The high-throughput and sensitive characterization of native proteins in biological samples is of increasing interest in multiple disciplines. Extractive electrospray ionization (EESI) forms ions of native proteins including lysozyme, α-chymotrypsin, myoglobin, human serum albumin, RNAse A and blood hemoglobin in extremely complex biosamples or PBS buffer solutions by softly depositing charges on the protein molecules. This method produces no significant conformational changes of the proteins in the ion formation process, and features direct detection of trace proteins present in biological matrices. The detection limit of low pmol L(-1) for lysozyme in untreated biological liquids such as human urine and tears was demonstrated using EESI mass spectrometry (MS), showing an attractive MS platform for the direct analysis of native proteins in actual biological samples.

Assessment of the nitrosylation process

To understand the principles and limits of the methodologies used for the measurement of S-nitrosylated proteins. Among methods for studying protein S-nitrosylation, chemoluminescence and biotin switch assay have rapidly gained popularity. However, recent findings have attempted to highlight potential pitfalls for these methods. Many assays for biological S-nitrosylated proteins are used near the limit of detection and pretreatment of the biological samples can modify the S-NO bond. These results suggest that additional controls are essential in order to identify S-nitrosylated proteins and results should be quantitatively validated using more than one methodology. Protein S-nitrosylation is emerging as a key mechanism by which nitric oxide regulates cell signalling. This review focuses on existing methodologies for the measurement of S-nitrosylated proteins in biological matrices and the potential pitfalls of each method.

Is ascorbate able to reduce disulfide bridges? A cautionary note

The biotin switch assay has recently been proposed as the eligible method to identify different S-nitrosated proteins in biological matrices. However, notwithstanding its wide application, a thorough validation of this method is still lacking. In particular, it has been suggested that ascorbate concentrations higher than 1 mM (as proposed in the original method) are needed since ascorbate reaction with S-nitrosothiols is slow. But the selectivity of ascorbate as a cleaving agent of the S–N bond under these conditions has not been well characterized. Our data indicate that ascorbate is able to reduce disulfide bridges of DTNB, cystine, cystinylglycine, glutathione disulfide, protein mixed disulfides and biotin-HPDP with pH and concentration dependent rates. Additionally, we tested the effect of indirect sunlight on ascorbate-mediated cleavage of both disulfides and S-nitrosothiols.

The application of ultra‐performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum

The detection and quantitation of apolipoproteins, important markers for coronary heart disease, in serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM) is reported. A tryptic digest of depleted human serum was analysed by nanoflow LC/MS/MS at a flow rate of 300 nL/min and several apolipoproteins (Apo), including Apo A1, A2, A4, C1, C2, C3, D, F and M, were successfully identified. The analysis of the same depleted serum digest by ultra-performance (UP)LC/MS/MS operating at 700 microL/min resulted in comparable sensitivity and selectivity to the nanoflow method, but with a dramatic ( approximately 20-fold) reduction in run time. The potential of UPLC/MS/MS for the rapid quantitation of proteins in biological matrices by representative tryptic peptides was further investigated using Apo A1 and its corresponding stable isotopically labelled tryptic AQUA peptide (DYVSQFEGSALGK). A set of serum-based Apo A1 calibrators from a clinical analyser kit were digested without depletion following the addition of the AQUA peptide and analysed using UPLC/MS/MS. A linear calibration curve was generated from peak area ratios to the labelled peptide with a coefficient of correlation of 0.9989. Standard curves were also generated for other apolipoproteins together with Apo B100, Apo E, lecithin cholesterol acyltransferase and albumin, which were also detected in the standards. The concentration of Apo A1 in five fresh undepleted human serum samples and a quality control (QC) sample were determined using both the UPLC/MS/MS method and a clinical analyser. Results were comparable and the quantitative study, involving 80 injections which took hours rather than days to complete, demonstrates the high-throughput potential of UPLC/MS/MS to quantify multiple serum proteins without the need for antibodies, and thus provide an alternative to the use of clinical analysers for serum protein biomarkers.

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography–protein digestion–liquid chromatography–mass spectrometry

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.