Observations from a decade of oligonucleotide bioanalysis by LC-MS.

There is a growing need for efficient bioanalysis of oligonucleotide therapeutics. This broad class of molecules presents numerous challenges relative to traditional small molecule therapeutics. Methodologies including ligand-binding assays or polymerase chain reaction may be fit-for-purpose in many instances, but liquid chromatography coupled to mass spectrometry (LC-MS) often delivers the best balance of sensitivity and selectivity. Over the last decade, we have engaged with many such molecules and derived insights into challenges and solutions. Herein, we provide four case studies illustrating challenges we have encountered. These issues include low or variable analyte recovery, poor resolution from related species, chromatographic abnormalities or challenging sensitivity. We present a summary of considerations, based on these experiences, to assist others working in the area.

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

The Origin-Adjusted Approach for Reliable Quantification of Endogenous Analytes in Mass Spectrometric Bioanalysis.

The reliably accurate and precise quantification of biomarkers is a priceless objective in the drug development and diagnostic arenas. To employ a technique that brings such reliability and furthermore involves a simpler, faster, and inexpensive regime would only underline the potential importance of the concept and technique. To the existing established approaches for biomarker quantification in bioanalytical LC-MS, surrogate matrix (SUR-M) and surrogate analyte (SUR-A), in this Letter we present an approach that fulfills the aforementioned advantages. The concept builds on the historic method of standard addition (SA), in which one source of biological matrix is spiked with analyte to form a calibration curve. With the SA curve back-calculated, the heart of this procedure is the subsequent adjustment of the intercept to zero, the origin, and using only the slope of the curve for interpolation giving calculated sample concentrations. In SA, the concentration axis intercept indicates the endogenous analyte concentration, and our zeroing of this is equivalent to removing the endogenous level. This key shift of the calculated line to the origin unveils our novel origin-adjusted (OA) approach. It enables use akin to a regular xenobiotic method, with no need to ultimately account for the endogenous analyte level in the control matrix used for calibrants. We present a comparison of OA against the control approach of SUR-M in a representative application for kynurenine and tryptophan in human plasma by LC-MS. A numerical performance analysis performed is demonstrative of equivalence between the two approaches for both analytes.

Sensitive LC-MS/MS quantification of unconjugated maytansinoid DM4 and its metabolite S-methyl-DM4 in human plasma.

Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.

High-sensitivity workflow for LC-MS-based analysis of GalNAc-conjugated oligonucleotides: a case study.

Aim: Mass-selective quantitation is a powerful attribute of LC-MS as a platform for bioanalysis. Here, a sensitive LC-MS approach has been validated for an oligonucleotide having chemical modifications (e.g., N-acetylgalactosamine [GalNAc] conjugated), to distinguish between the conjugated and unconjugated forms of the oligonucleotide, thereby enabling a nuanced view of the pharmacokinetic profile. Results: A high-sensitivity methodology for mass-specific measurement of AZD8233, a GalNAc-conjugated 16-mer oligonucleotide, using LLE-SPE with optimized LC conditions and detection of a low-mass fragment ion was successfully validated in the range of 0.20-100 ng/ml in human plasma. Conclusion: The AZD8233 LC-MS methodology adds valuable insight on the GalNAc linker's in vivo stability to the program and should be broadly applicable to oligonucleotides requiring high sensitivity and mass-selective measurement for quantitative discrimination from metabolites and endogenous interferences.

Development and validation of bioanalytical methods to support investigations of AZD9496 in the clinic.

Aim: AZD9496 is an oral nonsteroidal, potent and selective antagonist and degrader of ER-α. Two major active metabolites (M3 and M5 as diastereomers) were identified in humans. Methodology/results: Multianalyte, sensitive LC-MS/MS method in human plasma was developed and validated that overcame the challenges encountered. The method demonstrated acceptable precision, accuracy and selectivity for AZD9496 and two major metabolites. Incurred sample reanalysis was acceptable from evaluation in clinical studies, indicating adequate reproducibility. In addition, a urine method for AZD9496 was also developed and validated. Conclusion: Robust and sensitive LC-MS/MS assays for the quantitation of AZD9496 and two diastereomeric metabolites in human plasma and AZD9496 in human urine have been validated and successfully applied to clinical studies.

Whole blood stability in quantitative bioanalysis.

Establishing stability at all stages of a sample's lifespan is a critical part of performing regulated bioanalysis. For plasma assays, this includes the duration between when blood is drawn and when that blood is centrifuged to produce plasma. Here, we provide a discussion of current regulatory expectations around whole blood stability testing for LC-MS plasma assays, as well as the two primary experimental approaches utilized to assess whole blood stability. Next, we interrogated a large dataset of validated methods (1076 methods, the vast majority of which were for measurement of small molecules) to assess the correlation between whole blood and plasma stability profiles, finding them to be highly correlated. Finally, we summarize unique case studies; we have encountered during WB stability testing which offer lessons that may be broadly applicable.

Pharmacokinetics, metabolism, and excretion of nefopam, a dual reuptake inhibitor in healthy male volunteers.

1. The disposition of nefopam, a serotonin-norepinephrine reuptake inhibitor, was characterized in eight healthy male volunteers following a single oral dose of 75 mg [(14)C]-nefopam (100 µCi). Blood, urine, and feces were sampled for 168 h post-dose. 2. Mean (± SD) maximum blood and plasma radioactivity concentrations were 359 ± 34.2 and 638 ± 64.7 ngEq free base/g, respectively, at 2 h post-dose. Recovery of radioactive dose was complete (mean 92.6%); a mean of 79.3% and 13.4% of the dose was recovered in urine and feces, respectively. 3. Three main radioactive peaks were observed in plasma (metabolites M2 A-D, M61, and M63). Intact [(14)C]-nefopam was less than 5% of the total radioactivity in plasma. In urine, the major metabolites were M63, M2 A-D, and M51 which accounted for 22.9%, 9.8%, and 8.1% of the dose, respectively. An unknown entity, M55, was the major metabolite in feces (4.6% of dose). Excretion of unchanged [(14)C]-nefopam was minimal.

Serine effects on collision-induced dissociation and photodissociation of peptide cation radicals of the z+• -type.

The serine residue displays specific effects on the dissociations of peptide fragment cation-radicals of the z+• type which are produced by electron transfer dissociation. Energy-resolved collision-induced dissociation (ER-CID), time-resolved infrared multiphoton dissociation (TR-IRMPD), and single-photon UV photodissociation at 355 nm revealed several competitive dissociation pathways consisting of loss of OH radical, water, and backbone cleavages occurring at N-terminal and C-terminal positions relative to the serine residue. The activation modes using slow-heating and UV photon absorption resulted in different relative intensities of fragment ions. This indicated that the dissociations proceeded through several channels with different energy-dependent kinetics. The experimental data were interpreted with the help of electron structure calculations that provided fully optimized structures and relative energies for cis and trans amide isomers of the z4+• ions as well as isomerization, dissociation, and transition state energies. UV photon absorption by the z4+• ions was due to Cα-radical amide groups created by ETD that provided a new chromophore absorbing at 355 nm.

Competitive Hydrogen Atom Migrations Accompanying Cascade Dissociations of Peptide Cation-Radicals of the z+• Type.

We report a combined experimental and computational study of energy-resolved collision-induced dissociation (ER-CID) and time-resolved infrared multiphoton dissociation (TR-IRMPD) of z4 ions prepared by electron transfer dissociation of peptide (Ala-Ala-Asn-Ala-Arg + 2H)2+ ions. The z4 cation-radicals, •ANAR+, undergo competitive dissociations by backbone cleavage and loss of a CONH2 radical from the Asn side chain. The backbone cleavage proceeds by radical-assisted dissociation of the Asn Cα-CO bond, forming an x2 ion intermediate which rapidly dissociates by HNCO elimination to yield a stable z2 fragment ion, •AR+. The ER-CID and TR-IRMPD data were consistent with the consecutive nature of the backbone dissociation but showed different branching ratios for the two major fragmentations. The ER-CID data showed branching ratios 0.6-1.0 for the side-chain and backbone cleavages whereas the TR-IRMPD data showed an earlier onset for the latter dissociation. Computational analysis of the potential energy surface with density functional theory and ab initio calculations was carried out to provide structures and energies for the reactant ions as well as several intermediates, products, and transition states. Dissociation pathways for cis and trans amide conformers were distinguished and their energies were evaluated. The threshold dissociation energies for the backbone and side-chain dissociations were similar in accordance with the experimental ER-CID branching ratio. The TR-IRMPD data were interpreted by different absorbances of intermediates produced by hydrogen atom migrations along the dissociation pathways.

Case studies: the impact of nonanalyte components on LC-MS/MS-based bioanalysis: strategies for identifying and overcoming matrix effects.

Achieving sufficient selectivity in bioanalysis is critical to ensure accurate quantitation of drugs and metabolites in biological matrices. Matrix effects most classically refer to modification of ionization efficiency of an analyte in the presence of matrix components. However, nonanalyte or matrix components present in samples can adversely impact the performance of a bioanalytical method and are broadly considered as matrix effects. For the current manuscript, we expand the scope to include matrix elements that contribute to isobaric interference and measurement bias. These three categories of matrix effects are illustrated with real examples encountered. The causes, symptoms, and suggested strategies and resolutions for each form of matrix effects are discussed. Each case is presented in the format of situation/action/result to facilitate reading.

Activated ion ETD performed in a modified collision cell on a hybrid QLT-Oribtrap mass spectrometer.

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO2 laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety's high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

Multipurpose dissociation cell for enhanced ETD of intact protein species.

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

Tyrosine deprotonation yields abundant and selective backbone cleavage in peptide anions upon negative electron transfer dissociation and ultraviolet photodissociation.

Tyrosine deprotonation in peptides yields preferential electron detachment upon NETD or UVPD, resulting in prominent N-Cα bond cleavage N-terminal to the tyrosine residue. UVPD of iodo-tyrosine-modified peptides was used to generate localized radicals on neutral tyrosine side chains by homolytic cleavage of the C-I bond. Subsequent collisional activation of the radical species yielded the same preferential cleavage of the adjacent N-terminal N-Cα bond. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages are regularly observed for peptides when using high-pH mobile phases.

Cascade dissociations of peptide cation-radicals. Part 1. Scope and effects of amino acid residues in penta-, nona-, and decapeptides.

Amino acid residue-specific backbone and side-chain dissociations of peptide z ions in MS(3) spectra were elucidated for over 40 pentapeptides with arginine C-terminated sequences of the AAXAR and AAHXR type, nonapeptides of the AAHAAXX"AR and AAHAXAX"AR type, and AAHAAXX"AAR decapeptides. Peptide z(n) ions containing amino acid residues with readily transferrable benzylic or tertiary ß-hydrogen atoms (Phe, Tyr, His, Trp, Val) underwent facile backbone cleavages to form dominant z(n-2) or z(n-3) ions. These backbone cleavages are thought to be triggered by a side-chain ß-hydrogen atom transfer to the z ion C(α) radical site followed by homolytic dissociation of the adjacent C(α)-CO bond, forming x(n-2) cation-radicals that spontaneously dissociate by loss of HNCO. Amino acid residues that do not have readily transferrable ß-hydrogen atoms (Gly, Ala) do not undergo the z(n) → z(n-2) dissociations. The backbone cleavages compete with side-chain dissociations in z ions containing Asp and Asn residues. Side-chain dissociations are thought to be triggered by α-hydrogen atom transfers that activate the C(ß)-C(γ) or C(ß)-heteroatom bonds for dissociations that dominate the MS(3) spectra of z ions from peptides containing Leu, Cys, Lys, Met, Ser, Arg, Glu, and Gln residues. The Lys, Arg, Gln, and Glu residues also participate in γ-hydrogen atom transfers that trigger other side-chain dissociations.

Cascade dissociations of peptide cation-radicals. Part 2. Infrared multiphoton dissociation and mechanistic studies of z-ions from pentapeptides.

Dissociations of z(4) ions from pentapeptides AAXAR where X=H, Y, F, W, and V produce dominant z(2) ions that account for >50 % of the fragment ion intensity. The dissociation has been studied in detail by experiment and theory and found to involve several isomerization and bond-breaking steps. Isomerizations in z(4) ions proceed by amide trans→cis rotations followed by radical-induced transfer of a ß-hydrogen atom from the side chain, forming stable C(ß) radical intermediates. These undergo rate-determining cleavage of the C(α)-CO bond at the X residue followed by loss of the neutral AX fragment, forming x(2) intermediates. The latter were detected by energy-resolved resonant excitation collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) experiments. The x(2) intermediates undergo facile loss of HNCO to form z(2) fragment ions, as also confirmed by energy-resolved CID and IRMPD MS(4) experiments. The loss of HNCO from the x(2) ion from AAHWR is kinetically hampered by the Trp residue that traps the OCNH radical group in a cyclic intermediate.

Infrared multiphoton dissociation for quantitative shotgun proteomics.

We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.

Phosphorylation regulates human OCT4.

The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.

Implementing photodissociation in an Orbitrap mass spectrometer.

We modified a dual pressure linear ion trap Orbitrap to permit infrared multiphoton dissociation (IRMPD) in the higher energy collisional dissociation (HCD) cell for high resolution analysis. A number of parameters, including the pressures of the C-trap and HCD cell, the radio frequency (rf) amplitude applied to the C-trap, and the HCD DC offset, were evaluated to optimize IRMPD efficiency and maintain a high signal-to-noise ratio. IRMPD was utilized for characterization of phosphopeptides, supercharged peptides, and N-terminal modified peptides, as well as for top-down protein analysis. The high resolution and high mass accuracy capabilities of the Orbitrap analyzer facilitated confident assignment of product ions arising from IRMPD.

Activated-ion electron transfer dissociation improves the ability of electron transfer dissociation to identify peptides in a complex mixture.

Using a modified electron transfer dissociation (ETD)-enabled quadrupole linear ion trap (QLT) mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 strong cation exchanged (SCX) fractions of a LysC digest of human cell protein extract using ETD, collision-activated dissociation (CAD), and AI-ETD, we find that AI-ETD generates 13 405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7 968) and CAD (10 904). We also analyze 12 SCX fractions of a tryptic digest of human cell protein extract and find that ETD produces 6 234 PSMs, AI-ETD 9 130 PSMs, and CAD 15 209 PSMs. Compared to ETD with supplemental collisional activation (ETcaD), AI-ETD generates ∼80% more PSMs for the whole cell lysate digested with trypsin and ∼50% more PSMs for the whole cell lysate digested with LysC.