Analysis and Prediction of Chymotrypsin Substrate Preferences through Large Data Acquisition with Target-Free mRNA Display.

Oral delivery of peptide therapeutics is limited by degradation by gut proteases like chymotrypsin. Existing databases of peptidases are limited in size and do not enable systematic analyses of protease substrate preferences, especially for non-natural amino acids. Thus, stability optimization of hit compounds is time and resource intensive.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design.

Intact Mass Quantitation of Therapeutic Antibodies for Pharmacokinetic Studies Using Immuno-Purification.

The quantitation of therapeutic antibodies by mass spectrometry often utilizes a surrogate peptide approach following enzymatic digestion of the antibody. Although this approach has been widely adopted, it is labor intensive with limited throughput in most instances. In addition, this approach can pose challenges when attempting to infer details such as quantity and modification state of the intact analyte.

Improving the throughput of immunoaffinity purification and enzymatic digestion of therapeutic proteins using membrane-immobilized reagent technology.

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation.

Direct quantitation of therapeutic antibodies for pharmacokinetic studies using immuno-purification and intact mass analysis.

The quantitation of therapeutic antibodies by MS often utilizes a surrogate peptide approach. Recent enhancements in instrumentation and sample preparation have enabled quantitation by detection of the intact molecule using MS. A comparison of three methods for quantitative analysis of therapeutic monoclonal antibodies including analysis after deglycosylation, after hinge digestion and at the fully intact antibody level is reported.

Top-down LC-MS quantitation of intact denatured and native monoclonal antibodies in biological samples.

Aim: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody.

Results: Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation.

Quantitation of a Therapeutic Antibody in Serum Using Intact Sequential Affinity Capture, Trypsin Digestion, and LC-MS/MS.

Large molecule quantitation by LC-MS/MS commonly relies on bottom-up or so-called surrogate peptide measurements to infer the whole-molecule concentration. This can lead to questions about what is actually being measured in the assay (intact drug and/or other drug related material). An intact sequential affinity capture (ISAC) assay was developed utilizing two different immunoaffinity (IA) reagents.

Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK.

Aim: Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS.

Methods & Results: We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput.

Elevated AKAP12 in paclitaxel-resistant serous ovarian cancer cells is prognostic and predictive of poor survival in patients.

A majority of high-grade (HG) serous ovarian cancer (SOC) patients develop resistant disease despite high initial response rates to platinum/paclitaxel-based chemotherapy. We identified shed/secreted proteins in preclinical models of paclitaxel-resistant human HGSOC models and correlated these candidate proteins with patient outcomes using public data from HGSOC patients. Proteomic analyses of a HGSOC cell line secretome was compared to those from a syngeneic paclitaxel-resistant variant and from a line established from an intrinsically chemorefractory HGSOC patient.

Streamlining bottom-up protein identification based on selective ultraviolet photodissociation (UVPD) of chromophore-tagged histidine- and tyrosine-containing peptides.

We report a fast and highly efficient diazonium reaction that couples a nitroazobenzene chromophore to tyrosine and histidine residues, thus endowing peptides with high photoabsorption cross sections at 351 nm in the gas phase. Only the tagged peptides undergo ultraviolet photodissociation (UVPD) at 351 nm, as demonstrated for several Tyr- and His-containing peptides from protein digests. Additional selectivity is achieved by the integration of the UVPD-MS method with an in silico database search restricted to Tyr- and His-containing peptides.

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe.

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides.

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.

Enhancement of ultraviolet photodissociation efficiencies through attachment of aromatic chromophores.

Two N-terminal derivatization reagents containing aromatic chromophores, 4-sulfophenyl isothiocyanate (SPITC) and 4-methylphosphonophenyl isothiocyanate (PPITC), were used to increase the dissociation efficiencies of peptides upon ultraviolet photodissociation (UVPD) at 193 nm. The resulting UVPD spectra are dominated by C-terminal ions, including y, z, x, v, and w ions, and immonium ions. The attachment of the PPITC or SPITC groups leads to a reduction in the number and abundances of N-terminal ions because the added phosphonate or sulfonate functionalities result in neutralization of some of the N-terminal species, ones that might normally be singly protonated in the absence of the negatively charged sulfonate or phosphonate groups.

Enhanced electron transfer dissociation through fixed charge derivatization of cysteines.

Electron transfer dissociation (ETD) has proven to be a promising new ion activation method for proteomics applications due to its ability to generate c- and z-type fragment ions in comparison to the y- and b-type ions produced upon the more conventional collisional activation of peptides. However, low precursor charge states hinder the success of electron-based activation methods due to competition from nondissociative charge reduction and incomplete sequence coverage. In the present report, the reduction and alkylation of disulfide bonds prior to ETD analysis is evaluated by comparison of three derivatization reagents: iodoacetamide (IAM), N,N-dimethyl-2-chloro-ethylamine (DML), and (3-acrylamidopropyl)-trimethyl ammonium chloride (APTA).

Improved infrared multiphoton dissociation of peptides through N-terminal phosphonite derivatization.

A strategy for improving the sequencing of peptides by infrared multiphoton dissociation (IRMPD) in a linear ion trap mass spectrometer is described. We have developed an N-terminal derivatization reagent, 4-methylphosphonophenylisothiocyanate (PPITC), which allows the attachment of an IR-chromogenic phosphonite group to the N-terminus of peptides, thus enhancing their IRMPD efficiencies. After the facile derivatization process, the PPITC-modified peptides require shorter irradiation times for efficient IRMPD and yield extensive series of y ions, including those of low m/z that are not detected upon traditional CID.

Chromogenic cross-linker for the characterization of protein structure by infrared multiphoton dissociation mass spectrometry.

We have developed a new IR chromogenic cross-linker (IRCX) to aid in rapidly distinguishing cross-linked peptides from unmodified species in complex mixtures. By incorporating a phosphate functional group into the cross-linker, one can take advantage of its unique IR absorption properties, affording selective infrared multiphoton dissociation (IRMPD) of the cross-linked peptides. In a mock mixture of unmodified peptides and IRCX-cross-linked peptides (intramolecularly and intermolecularly cross-linked), only the peptides containing the IRCX modification were shown to dissociate upon exposure to 50 ms of 10.