Quantifying KRAS G12C Covalent Drug Inhibitor Activity in Mouse Tumors Using Mass Spectrometry.

The growing opportunities recognized for covalent drug inhibitors, like KRAS G12C inhibitors, are driving the need for mass spectrometry methods that can quickly and robustly measure therapeutic drug activity in vivo for drug discovery research and development. Effective front-end sample preparation is critical for proteins extracted from tumors but is generally labor intensive and impractical for large sample numbers typical in pharmacodynamic (PD) studies. Herein, we describe an automated and integrated sample preparation method for the measurement of activity levels of KRAS G12C drug inhibitor alkylation from complex tumor samples involving high throughput detergent removal and preconcentration followed by quantitation using mass spectrometry. We introduce a robust assay with an average intra-assay coefficient of variation (CV) of 4% and an interassay CV of 6% obtained from seven studies, enabling us to understand the relationship between KRAS G12C target occupancy and the therapeutic PD effect from mouse tumor samples. Further, the data demonstrated that the drug candidate GDC-6036, a KRAS G12C covalent inhibitor, shows dose-dependent target inhibition (KRAS G12C alkylation) and MAPK pathway inhibition, which correlate with high antitumor potency in the MIA PaCa-2 pancreatic xenograft model.

Corrigendum to "Analytical validation of protein biomarkers for risk of spontaneous preterm birth" [Clin. Mass Spectrom. 3 (2017) 25-38].

[This corrects the article DOI: 10.1016/j.clinms.2017.06.002.].

Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection.

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Analytical validation of protein biomarkers for risk of spontaneous preterm birth.

Presented are the validation results of a second-generation assay for determining the relative abundances of two protein biomarkers found in maternal serum that predict an individual's risk of spontaneous preterm birth. The sample preparation workflow is complex, consisting of immuno-depletion of high-abundance serum proteins, tryptic digestion of the immuno-depleted fraction to generate surrogate peptide analytes, and detection by tandem mass spectrometry. The method was determined to be robust on observation of the following characteristics: classifier peptide detection precision was excellent; results were accurate when compared to a reference method; results were linear over a clinically relevant range; the limits of quantitation encompassed the range of expected results; and the method demonstrated analytical specificity and resilience to differences in patient serum and common endogenous interferents.

Host Protein Biomarkers Identify Active Tuberculosis in HIV Uninfected and Co-infected Individuals.

Biomarkers for active tuberculosis (TB) are urgently needed to improve rapid TB diagnosis. The objective of this study was to identify serum protein expression changes associated with TB but not latent Mycobacterium tuberculosis infection (LTBI), uninfected states, or respiratory diseases other than TB (ORD). Serum samples from 209 HIV uninfected (HIV(-)) and co-infected (HIV(+)) individuals were studied. In the discovery phase samples were analyzed via liquid chromatography and mass spectrometry, and in the verification phase biologically independent samples were analyzed via a multiplex multiple reaction monitoring mass spectrometry (MRM-MS) assay. Compared to LTBI and ORD, host proteins were significantly differentially expressed in TB, and involved in the immune response, tissue repair, and lipid metabolism. Biomarker panels whose composition differed according to HIV status, and consisted of 8 host proteins in HIV(-) individuals (CD14, SEPP1, SELL, TNXB, LUM, PEPD, QSOX1, COMP, APOC1), or 10 host proteins in HIV(+) individuals (CD14, SEPP1, PGLYRP2, PFN1, VASN, CPN2, TAGLN2, IGFBP6), respectively, distinguished TB from ORD with excellent accuracy (AUC = 0.96 for HIV(-) TB, 0.95 for HIV(+) TB). These results warrant validation in larger studies but provide promise that host protein biomarkers could be the basis for a rapid, blood-based test for TB.

Cation recombination energy/coulomb repulsion effects in ETD/ECD as revealed by variation of charge per residue at fixed total charge.

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) experiments in electrodynamic ion traps operated in the presence of a bath gas in the 1-10 mTorr range have been conducted on a common set of doubly protonated model peptides of the form X(AG)nX (X = lysine, arginine, or histidine, n = 1, 2, or 4). The partitioning of reaction products was measured using thermal electrons, anions of azobenzene, and anions of 1,3-dinitrobenzene as reagents. Variation of n alters the charge per residue of the peptide cation, which affects recombination energy. The ECD experiments showed that H-atom loss is greatest for the n = 1 peptides and decreases as n increases. Proton transfer in ETD, on the other hand, is expected to increase as charge per residue decreases (i.e., as n increases). These opposing tendencies were apparent in the data for the K(AG)nK peptides. H-atom loss appeared to be more prevalent in ECD than in ETD and is rationalized on the basis of either internal energy differences, differences in angular momentum transfer associated with the electron capture versus electron transfer processes, or a combination of the two. The histidine peptides showed the greatest extent of charge reduction without dissociation, the arginine peptides showed the greatest extent of side-chain cleavages, and the lysine peptides generally showed the greatest extent of partitioning into the c/z•-product ion channels. The fragmentation patterns for the complementary c- and z•-ions for ETD and ECD were found to be remarkably similar, particularly for the peptides with X = lysine.

Gas-phase intramolecular protein crosslinking via ion/ion reactions: ubiquitin and a homobifunctional sulfo-NHS ester.

Gas-phase intra-molecular crosslinking of protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of a homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin and crosslinker monoanions produces a stable, charge-reduced complex. Covalent crosslinking is indicated by the consecutive loss of 2 molecules of sulfo-NHS under ion trap collisional activation conditions. Covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The presence of two unprotonated reactive sites within the distance constraint of the crosslinker is required for successful crosslinking. The ability to covalently crosslink is, therefore, sensitive to protein charge state. As the charge state increases, fewer reactive sites are available and protein structure is more likely to become extended because of intramolecular electrostatic repulsion. At high charge states, the reagent shows little evidence for covalent crosslinking but does show evidence for 'electrostatic crosslinking' in that the binding of the sulfonate groups to the protein is sufficiently strong that backbone cleavages are favored over reagent detachment under ion trap collisional activation conditions.

Gas-phase conjugation to arginine residues in polypeptide ions via N-hydroxysuccinimide ester-based reagent ions.

Gas-phase conjugation to unprotonated arginine side-chains via N-hydroxysuccinimide (NHS) esters is demonstrated through both charge reduction and charge inversion ion/ion reactions. The unprotonated guanidino group of arginine can serve as a strong nucleophile, resulting in the facile displacement of NHS from NHS esters with concomitant covalent modification of the arginine residue. This reactivity is analogous to that observed with unprotonated primary amines such as the N-terminus or ε-amino group of lysine. In solution, however, the arginine residues tend to be protonated at pH values low enough to prevent hydrolysis of NHS esters, which would render them relatively unreactive with NHS esters. This work demonstrates novel means for gas-phase conjugation to arginine side chains in polypeptide ions.

Solution versus gas-phase modification of peptide cations with NHS-ester reagents.

A comparison between solution and gas phase modification of primary amine sites in model peptide cations with N-hydroxysuccinimide (NHS) ester reagents is presented. In all peptides, the site of modification in solution was directed to the N-terminus by conducting reactions at pH=5, whereas for the same peptides, a lysine residue was preferentially modified in the gas phase. The difference in pKa values of the N-terminus and ε-amino group of the lysine allows for a degree of control over sites of protonation of the peptides in aqueous solution. With removal of the dielectric and multiple charging of the peptide ions in the gas phase, the accommodation of excess charge can affect the preferred sites of reaction. Interaction of the lone pair of the primary nitrogen with a proton reduces its nucleophilicity and, as a result, its reactivity towards NHS-esters. While no evidence for reaction of the N-terminus with sulfo-NHS-acetate was noted in the model peptide cations, a charge inversion experiment using bis[sulfosuccinimidyl] suberate, a cross-linking reagent with two sulfo-NHS-ester functionalities, showed modification of the N-terminus. Hence, an unprotonated N-terminus can serve as a nucleophile to displace NHS, which suggests that its lack of reactivity with the peptide cations is likely due to the participation of the N-terminus in solvating excess charge.

Cleavage of Multiple Disulfide Bonds in Insulin via Gold Cationization and Collision-induced Dissociation.

Intact bovine insulin, with its two chains linked via two disulfide linkages, has been used as a model system to study the incorporation of one or more gold cations as means for facilitating the cleavage of multiple disulfide bonds in a tandem mass spectrometry experiment. Gas-phase ion/ion reactions involving Au(I)Cl(2) (-) or Au(III)Cl(4) (-) were used to incorporate either one or two gold cations into multiply-protonated insulin cations, followed by ion trap collision-induced dissociation (CID) of the products. The incorporation of a single gold cation followed by CID showed little evidence for disulfide bond cleavage. Rather, the CID spectra were similar to those acquired for the same charge state with only excess protons present. However, the incorporation of two gold cations, regardless of oxidation state, resulted in efficient cleavage of the disulfide bonds connecting the two chains of insulin. Furthermore, ion trap CID of the insulin complexes containing two gold cations showed more sequence information compared to the complexes containing only one gold cation or no gold cations. The partitioning of the gold cations between the two chains upon CID proved to be largely asymmetric, as both gold cations tended to stay together. There appeared to be a slight preference for both gold cations to partition into the B-chain. However, the relatively low contribution from single chain ions with only one gold ion suggests a degree of cooperativity in the overall mechanism for separation of the two chains.

Intra- and inter-molecular cross-linking of peptide ions in the gas phase: reagents and conditions.

Intra-molecular and inter-molecular cross-linking of protonated polypeptide ions in the gas phase via ion/ion reactions have been demonstrated using N-hydroxysulfosuccinimide (sulfo-NHS)- based reagent anions. The initial step in the ion/ion reaction involves the formation of a long-lived complex between the peptide and reagent, which is a prerequisite for the covalent bioconjugation chemistry. The sulfonate groups on the NHS rings of the homo-bifunctional cross-linking reagents have high affinity for the protonated sites in the peptide and, therefore, facilitate the long-lived complex formation. In addition to the formation of a long-lived chemical complex, intra-molecular cross-linking also requires two unprotonated primary amine sites within a molecule where the covalent modification takes place. Alternatively, inter-molecular cross-linking demands the availability of one neutral primary amine site in each of the two peptides that are being cross-linked. Nucleophilic displacement of two sulfo-NHS groups by the amine functionalities in the peptide is a signature of the covalent cross-linking chemistry in the gas phase. Upon removal of the two sulfo-NHS groups, two amide bonds are formed between an unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent.

Ion/neutral, ion/electron, ion/photon, and ion/ion interactions in tandem mass spectrometry: do we need them all? Are they enough?

A range of strategies and tools have been developed to facilitate the determination of primary structures of analyte molecules of interest via tandem mass spectrometry (MS/MS). The two main factors that determine the primary structural information present in an MS/MS spectrum are the type of ion generated from the analyte molecule and the dissociation method. The ion type subjected to dissociation is determined by the ionization method/conditions and ion transformation processes that might take place after initial gas-phase ion formation. Furthermore, the range of analyte-related ion types can be expanded via derivatization reactions prior to mass spectrometry. Dissociation methods include those that simply alter the population of internal states of the mass-selected ion (i.e., activation methods like collision-induced dissociation) as well as processes that rely on the transformation of the ion type prior to dissociation (e.g., electron capture dissociation). A variety of ion interactions have been studied for the purpose of ion dissociation and ion transformation, including ion/neutral, ion/photon, ion/electron, and ion/ion interactions. A wide range of phenomena have been observed, many of which have been explored/developed as means for structural analysis. The techniques arising from these phenomena are discussed within the context of the elements of structural determination in tandem mass spectrometry: ion-type definition and dissociation. Unique aspects of the various ion interactions are emphasized along with any barriers to widespread implementation.

Covalent modification of gaseous peptide ions with N-hydroxysuccinimide ester reagent ions.

Covalent modification of primary amine groups in multiply protonated or deprotonated polypeptides in the gas phase via ion/ion reactions is demonstrated using N-hydroxysuccinimide (NHS) esters as the modifying reagents. During the ion/ion reaction, the peptide analyte ions and the NHS or sulfo-NHS based reagent form a long-lived complex, which is a prerequisite for the covalent modification chemistry to occur. Ion activation of the peptide-reagent complex results in a neutral NHS or sulfo-NHS molecule loss, which is a characteristic signature of covalent modification. As the NHS or sulfo-NHS group leaves, an amide bond is formed between a free, unprotonated, primary amine group of a lysine side chain in the peptide and the carboxyl group in the reagent. Subsequent activation of the NHS or sulfo-NHS loss product ions results in sequence informative fragment ions containing the modification. The N-terminus primary amine group does not make a significant contribution to the modification process; this behavior has also been observed in solution phase reactions. The ability to covalently modify primary amine groups in the gas phase with N-hydroxysuccinimide reagents opens up the possibility of attaching a wide range of chemical groups to gaseous peptides and proteins and also for selectively modifying other analytes containing free primary amine groups.

Dissociation of disulfide-intact somatostatin ions: the roles of ion type and dissociation method.

The dissociation chemistry of somatostatin-14 was examined using various tandem mass spectrometry techniques including low-energy beam-type and ion trap collision-induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide-gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin-14 is present within a loop defined by the disulfide linkage between Cys-3 and Cys-14. The generation of readily interpretable sequence-related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH(2)-S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H](2+) species. The products were generated by a combination of S-S bond cleavage and amide bond cleavage. ETD of the [M+3H](3+) ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S-S bond and an N-C(alpha) bond can be cleaved following a single electron transfer reaction.