Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments From the ‡Broad Institute of MIT and Harvard, Cambridge, Massachusetts; §Eli

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.

Quantitative bioanalysis by direct nanoelectrospray infusion coupled to tandem mass spectrometry has been achieved using an automated liquid sampler integrated with an array of microfabricated electrospray nozzles allowing rapid, serial sample introduction (1 min/ sample). Standard curves prepared in human plasma for verapamil (r2 = 0.999) and its metabolite norverapamil (r2 = 0.998) were linear over a range of 2.5-500 ng/ mL. Based on the observed precision and accuracy, a lower limit of quantitation of 5 ng/mL was assigned for both analytes. Sample preparation consisted of protein precipitation with an organic solvent containing the structural analogue gallopamil as an internal standard. Protein precipitation was selected both to maximize throughput and to test the robustness of direct nanoelectrospray infusion. Aliquots of supernatant (10 pL) were transferred to the back plane of the chip using disposable, conductive pipet tips for direct infusion at a flow rate of 300 nL/min. Electrospray ionization occurred from the etched nozzles (30-microm o.d.) on the front of the chip, initiated by a voltage applied to the liquid through the pipet tip. The chip was positioned near the API sampling orifice of a triple quadrupole mass spectrometer, which was operated in selected reaction monitoring mode. Results are presented that document the complete elimination of system carry-over, attributed to lack of a redundant fluid path. This technology offers potential advantages for MS-based screening applications in drug discovery by reducing the time for methods development and sample analysis.

Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.

LC/MS/MS based bioanalysis using atmospheric pressure ionization (API)-style interfaces has now been applied for over a decade. This technology, which initially found application for clinical bioanalysis, is now firmly established as the primary bioanalytical tool for ADME studies related to drug discovery and lead optimization (LO). This review focuses on recent advances in LC/MS/MS based bioanalysis in support of drug discovery and LO. The initial part of the article reviews the principal components of LC/MS/MS bioanalysis: sample preparation, chromatography, ionization and mass analysis. In each section, factors affecting high throughput bioanalysis are addressed. Because of the importance of on-line column switching methods to discovery bioanalysis, the section on sample preparation is divided into off-line and on-line approaches. In addition, the discussion of chromatography is limited to reversed phase liquid chromatography with emphasis given to the trend towards high-flow gradient elution techniques. The latter part of the review focuses on considerations for experimental design. In this section, pooling methods such as cassette dosing are discussed along with more highly integrated strategies linking bioanalysis with protocol generation and sample collection. The article concludes by briefly reviewing factors, which affect bioanalytical precision and accuracy, such as ion suppression, analyte stability and metabolite interference.

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N- (4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC 50 ϭ 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleeppromoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.

For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.