Absolute Quantification of RNA or DNA Using Acid Hydrolysis and Mass Spectrometry.

Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.

Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Amino Acid Analysis.

The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for assigning concentration values in SRM deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS-the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.

Quantification of cardiac troponin I in human plasma by immunoaffinity enrichment and targeted mass spectrometry.

Quantification of cardiac troponin I (cTnI), a protein biomarker used for diagnosing myocardial infarction, has been achieved in native patient plasma based on an immunoaffinity enrichment strategy and isotope dilution (ID) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The key steps in the workflow involved isolating cTnI from plasma using anti-cTnI antibody coupled to magnetic nanoparticles, followed by an enzymatic digestion with trypsin. Three tryptic peptides from cTnI were monitored and used for quantification by ID-LC-MS/MS via multiple reaction monitoring (MRM). Measurements were performed using a matrix-matched calibration system. NIST SRM 2921 Human Cardiac Troponin Complex acted as the calibrant and a full-length isotopically labeled protein analog of cTnI was used as an internal standard. The method was successfully demonstrated on five patient plasma samples, with cTnI concentrations measuring between 4.86 µg/L and 11.3 µg/L (signifying moderate myocardial infarctions). LC-MS/MS measurement precision was validated by three unique peptides from cTnI and two MRM transitions per peptide. Relative standard deviation (CV) from the five plasma samples was determined to be ≤14.3%. This study has demonstrated that quantification of cTnI in native plasma from myocardial infarction patients can be achieved based on an ID-LC-MS/MS method. The development of an ID-LC-MS/MS method for cTnI in plasma is a first step for future certification of matrix-based reference materials, which may be used to help harmonize discordant cTnI clinical assays. Graphical abstract A schematic of the workflow for measuring cardiac troponin I (cTnI), a low-abundant protein biomarker used for diagnosing myocardial infarction, in human plasma by isotope-dilution LC-MS/MS analysis.

Quantification of antibody coupled to magnetic particles by targeted mass spectrometry.

Quantifying the amount of antibody on magnetic particles is a fundamental, but often overlooked step in the development of magnetic separation-based immunoaffinity enrichment procedures. In this work, a targeted mass spectrometry (MS)-based method was developed to directly measure the amount of antibody covalently bound to magnetic particles. Isotope-dilution liquid chromatography-tandem MS (ID-LC-MS/MS) has been extensively employed as a gold-standard method for protein quantification. Here, we demonstrate the utility of this methodology for evaluating different antibody coupling processes to magnetic particles of different dimensions. Synthesized magnetic nanoparticles and pre-functionalized microparticles activated with glutaraldehyde or epoxy surface groups were used as solid supports for antibody conjugation. The key steps in this quantitative approach involved an antibody-magnetic particle coupling process, a wash step to remove unreacted antibody, followed by an enzymatic digestion step (in situ with the magnetic particles) to release tryptic antibody peptides. Our results demonstrate that nanoparticles more efficiently bind antibody when compared to microparticles, which was expected due to the larger surface area per unit mass of the nanoparticles compared to the same mass of microparticles. This quantitative method is shown to be capable of accurately and directly measuring antibody bound to magnetic particles and is independent of the conjugation method or type of magnetic particle. Graphical Abstract Schematic illustration of the isotope-dilution mass spectrometry-based workflow to directly measure antibody bound to magnetic particles (MP).

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Production, Purification, and Characterization of ¹5N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements.

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.

Addiction to MTH1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry.

MTH1 protein sanitizes the nucleotide pool so that oxidized 2'-deoxynucleoside triphosphates (dNTPs) cannot be used in DNA replication. Cancer cells require MTH1 to avoid incorporation of oxidized dNTPs into DNA that results in mutations and cell death. Inhibition of MTH1 eradicates cancer, validating MTH1 as an anticancer target. By overexpressing MTH1, cancer cells may mediate cancer growth and resist therapy. To date, there is unreliable evidence suggesting that MTH1 is increased in cancer cells, and available methods to measure MTH1 levels are indirect and semi-quantitative. Accurate measurement of MTH1 in disease-free tissues and malignant tumors of patients may be essential for determining if the protein is truly upregulated in cancers, and for the development and use of MTH1 inhibitors in cancer therapy. Here, we present a novel approach involving liquid chromatography-isotope-dilution tandem mass spectrometry to positively identify and accurately quantify MTH1 in human tissues. We produced full length (15)N-labeled MTH1 and used it as an internal standard for the measurements. Following trypsin digestion, seven tryptic peptides of both MTH1 and (15)N-MTH1 were identified by their full scan and product ion spectra. These peptides provided a statistically significant protein score that would unequivocally identify MTH1. Next, we identified and quantified MTH1 in human disease-free breast tissues and malignant breast tumors, and in four human cultured cell lines, three of which were cancer cells. Extreme expression of MTH1 in malignant breast tumors was observed, suggesting that cancer cells are addicted to MTH1 for their survival. The approach described is expected to be applicable to the measurement of MTH1 levels in malignant tumors vs. surrounding disease-free tissues in cancer patients. This attribute may help develop novel treatment strategies and MTH1 inhibitors as potential drugs, and guide therapies.

Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.

Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.

A quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.

A mass spectrometry-based antibody selection procedure was developed to evaluate optimal 'capture' monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K(d)) were determined for 'bound mAb-antigen' vs. 'unbound antigen' using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal 'capture' mAbs were determined through evaluation of relative K(d) constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K(d) constants as determined using NIST Standard Reference Material (SRM) 2921 - Human Cardiac Troponin Complex. This ID LC-MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification.

Comparison of orthogonal liquid and gas chromatography-mass spectrometry platforms for the determination of amino acid concentrations in human plasma.

Concentrations of amino acids in a human plasma pool were determined using four independent quantification methods. Orthogonal separation schemes (LC, GC, or GC x GC) and detection systems (triple quadrupole or time-of-flight mass spectrometry) are shown to demonstrate excellent consistency among platforms for quantifying 18 amino acids in NIST Standard Reference Material (SRM) 1950 Metabolites in Human Plasma using a well-characterized isotope dilution (ID) quantification method. Measured levels were consistent with reference values in plasma from the literature. Individual amino acid concentrations in plasma varied by over an order of magnitude ranging from 1.83 microg/g to 28.0 microg/g (7.78 micromol/L to 321 micromol/L). Average variability (coefficient of variation) between experimental amino acid concentrations (excluding cysteine) among all methods was 6.3%. Certified mass fraction values for amino acids in NIST SRM 1950 will be established from statistically weighted means of all experimental results.

Identification and characterization of endogenous Langerin ligands in murine extracellular matrix.

Langerin is a C-type lectin that is expressed by Langerhans cells (LC) and related immune cells, and believed to play an important role in antigen recognition and uptake. To determine if Langerin has endogenous ligands, we generated S protein binding, bacterial recombinant, mouse soluble Langerin, and utilized it as a probe. Recombinant soluble Langerin did not bind to lymph node or spleen cells, or keratinocytes as assessed via flow cytometry. However, Langerin did bind to surfaces of primary skin fibroblasts and NIH3T3 cells. "Ligand blotting" of fibroblast membrane-enriched fractions with Langerin revealed reproducible binding to 140 and 240 kDa proteins resolved in reduced denaturing gels. Characterization of these proteins using mass spectrometry suggested types I and III procollagen and fibronectin as candidate ligands. Langerin bound to type I procollagen that was immunoprecipitated from fibroblast lysates, but did not bind to fibronectin that was immunoprecipitated from fibroblast-conditioned media or mouse plasma fibronectin. These results indicate that Langerin selectively interacts with at least one ligand in extracellular matrix (type I procollagen). Langerin may have an unanticipated role in cell-matrix interactions that modulate LC development, localization, or function.

Analysis of albumin-associated peptides and proteins from ovarian cancer patients.

BACKGROUND: Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. METHODS: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. RESULTS: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. CONCLUSION: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Biomarker amplification by serum carrier protein binding.

Mass spectroscopic analysis of the low molecular mass (LMM) range of the serum/plasma proteome is a rapidly emerging frontier for biomarker discovery. This study examined the proportion of LMM biomarkers, which are bound to circulating carrier proteins. Mass spectroscopic analysis of human serum following molecular mass fractionation, demonstrated that the majority of LMM biomarkers exist bound to carrier proteins. Moreover, the pattern of LMM biomarkers bound specifically to albumin is distinct from those bound to non-albumin carriers. Prominent SELDI-TOF ionic species (m/z 6631.7043) identified to correlate with the presence of ovarian cancer were amplified by albumin capture. Several insights emerged: a) Accumulation of LMM biomarkers on circulating carrier proteins greatly amplifies the total serum/plasma concentration of the measurable biomarker, b) The total serum/plasma biomarker concentration is largely determined by the carrier protein clearance rate, not the unbound biomarker clearance rate itself, and c) Examination of the LMM species bound to a specific carrier protein may contain important diagnostic information. These findings shift the focus of biomarker detection to the carrier protein and its biomarker content.