Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Enhancing the accuracy of measurement of small molecule organic biomarkers.

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.

Quantification of growth hormone in serum by isotope dilution mass spectrometry.

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u(c)) of 3.0%, and a limit of quantification (LOQ) of 1.7 microg/L when quantifying T6 as a GH-derived fragment, whereas recovery=100.7%, SD=2.4%, u(c)=2.5%, and LOQ=2.7 microg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.

Protein quantification by isotope dilution mass spectrometry of proteolytic fragments: cleavage rate and accuracy.

The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U = 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.

Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins.

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.

Determination of HbA2 by quantitative bottom-up proteomics and isotope dilution mass spectrometry.

BACKGROUND: Poor comparability between laboratories is often observed in the measurement of HbA2. A measurement procedure of higher metrological order is needed for value assignment to a reference material that shall be used as primary calibrator. METHOD: A reference measurement procedure has been developed based on isotope dilution mass spectrometry (IDMS). The α- and δ- subunits are quantified by signature peptides released by tryptic digestion of a 25 µL-blood sample. Full length U-15N-labeled HbA0 and HbA2 are used as internal standards and added to the sample at concentrations closely matching the levels of the natural forms in blood. By this, an improvement in precision could be achieved with respect to previous mass-spectrometry based methods. RESULTS: Recovery of HbA2 added to a blood sample was within 102.6-105.2%. Repeatability and within-laboratory imprecision was <2.0% for two blood samples containing HbA2 at a low and a high fraction. Total combined measurement uncertainty is estimated as 5.5%. Good agreement (r = 0.998) of results was obtained in a comparison of two laboratories using the described IDMS procedure. There is good correlation between commercial analytical systems and IDMS (r = 0.975-0.989). Some of the platforms provide significantly biased results, however, which potentially could be mitigated by reference to IDMS. CONCLUSION: IDMS holds a promise to be suitable as a reference measurement procedure for standardization of HbA2-measurements in laboratory medicine.

Growth hormone isoform-differential mass spectrometry for doping control purposes.

Mass spectrometry (MS) allows for monitoring growth hormone (GH) isoform compositions at high specificity. It is demonstrated that this capability can be used to reliably detect alterations as elicited by (putative) doping with 22 kDa-GH. Sample treatment consists of enzymatic protein cleavage, followed by 2-step liquid chromatography clean-up, prior to analysis by MS. The protocol does not depend on antibodies for analyte extraction at any stage. Therefore, MS opens an opportunity for independent confirmation, if combined with the antibody-based isoform differential test presently used in practice. To check the fitness-for-purpose of this concept, GH-free serum was spiked with pure 22 kDa- and 20 kDa-GH covering a representative range of concentrations (0.5-9.4 µg/L), while the 22kDa fraction was within a range of  80%-85%, or at 100%, the latter simulating an administration of 22 kDa-GH. Mean deviation of 22 kDa-fractions found was within less than 3% for samples with total GH>= 1 µg/L . Beyond this, results by antibody-free isoform-differential MS, as described, were in line with those of the World Anti-Doping Agency-approved antibody-based test for 18 native sera and 3 positive controls. In this context, relating 22 kDa-GH to total-GH rather than 22 kDa+20 kDa was considered as an alternative strategy to earlier approaches. However, 20 kDa-GH as an additional measurand, next to 22 kDa- and total-GH, provides useful extra information, as it directly indicates the presence or absence of a non-22 kDa-GH form.

Engineering monolayer poration for rapid exfoliation of microbial membranes.

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

Determination of Cystatin C in human serum by isotope dilution mass spectrometry using mass overlapping peptides.

We propose a peptide-based isotope dilution mass spectrometry approach for Cystatin C determination in human serum samples, a clinical marker for renal status for which backup by a mass spectrometry based primary method has been missing so far. In contrast to common protocols, the isotope labelled version of the proteotypic signature peptide is designed such as keeping the isotopic difference as little as possible with respect to the peptide released from the protein. Peptides labelled in two (13)C atoms are added to the serum samples just before proteolysis. After two steps of chromatographic purification the sample is measured by selected reaction monitoring using a LC-MS/MS. Resolution of the first quadrupole is reduced to transmit the whole parent ion cluster to the collision cell for monitoring accurate isotopic distributions of the molecular fragments. Molar fractions of labelled and natural abundance peptides are directly obtained from the experimental mass spectra of the in-cell fragment ions. Thus, the natural abundance protein concentration is obtained from the fragment-ion spectrum of the sample without resorting to extra calibration runs. Applicability of the approach is demonstrated by the measurement of the serum concentration of Cystatin C in Reference Material ERM R-DA471/IFCC and real samples. BIOLOGICAL SIGNIFICANCE: Cystatin C is used as an alternative marker instead of, or in combination with creatinine for non-invasive determination of glomerular filtration rates. Advantages advocating in favour of Cystatin C in diagnosis of chronic kidney diseases are the lower variability of its serum level and, particularly, virtual independence on sex, age and muscle mass. However, in order to capitalize, accuracy of measurement has to be in proportion with the predictive power of the marker. Though there are label-free methods available for screening purposes or high-throughput analysis, achieving high levels of reliability and accuracy in quantitative proteomics takes reference to isotope labelled materials. Present routine assays (mainly nephelometry, turbidimetry and ligand-binding assays) are known to leave improvement to be desired in that respect. Absolute quantification based on enzymatic signature-peptides provides a method principle establishing traceability to the International System of Units on the level of primary methods. The kind of technique is capable, by this way, of high accuracy value-assignment to matrix materials needed for calibration of present routine assays, where not completely replacing them. Cystatin C measurement by isotope dilution mass spectrometry is developed in this study with the aim of making available this tool to support diagnostics of kidney function in the same way.

Mass spectrometry - an alternative in growth hormone measurement.

Growth hormone (GH) constitutes a set of closely related protein isoforms. In clinical practice, the disagreement of test results between commercially available ligand-binding assays is still an ongoing issue, and incomplete knowledge about the particular function of the different forms leaves an uncertainty of what should be the appropriate measurand. Mass spectrometry is promising to be a way forward. Not only is it capable of providing SI-traceable reference values for the calibration of current GH-tests, but it also offers an independent approach to highly reliable mass-selective quantification of individual GH-isoforms. This capability may add to reliability in doping control too. The article points out why and how.

High sensitivity mass spectrometric quantification of serum growth hormone by amphiphilic peptide conjugation.

Amphiphilic peptide conjugation affords a significant increase in sensitivity with protein quantification by electrospray-ionization mass spectrometry. This has been demonstrated for human growth hormone (GH) in serum using N-(3-iodopropyl)-N,N,N-dimethyloctylammonium iodide as derivatizing reagent. The signal enhancement achieved is up to a factor of 5-6 and enables extension of the applicable concentration range down to the very low concentrations (≤ 1.0 µg/L) as encountered with clinical glucose suppression tests for patients with acromegaly. The method has been validated using a set of serum samples spiked with known amounts of recombinant 22 kDa GH in the range of 0.48 to 7.65 µg/L. The coefficient of variation (CV) calculated based on the deviation of results from the expected concentrations was 3.5%. The limit of detection (LoD) was determined as 0.1 µg/L and the limit of quantification (LoQ) as 0.4 µg/L. The potential of the method as a tool in clinical practice has been demonstrated with patient samples of about 1 µg/L.

Surface-enhanced Raman scattering based approach for quantitative determination of creatinine in human serum.

A novel surface-enhanced Raman scattering (SERS) based approach for the quantitative determination of creatinine in human serum is described. Using isotopically labeled (2-13C, 2,3-15N2) creatinine as internal standard, SERS acquires the character of a ratio method that works similar to the well-established isotope dilution techniques. In conjunction with multivariate data analysis, the method was successfully applied for quantifying creatinine at clinically relevant levels and below. A partial least-squares regression model was generated from a set of 87 calibration spectra covering the full range of mole fractions of neat creatinine. The prediction performance of the model was thereafter validated with independent reference samples giving a standard deviation of less than 2%. Finally, a conditioning procedure to prepare real serum samples for SERS-based creatinine analysis was worked out and validated. Measured serum creatinine concentrations are within 3% of the values obtained from gas chromatography/isotope dilution mass spectrometry on the same serum starting material.