Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody.

Aspartic acid (Asp) to isoaspartic acid (isoAsp) isomerization in therapeutic monoclonal antibodies (mAbs) and other biotherapeutics is a critical quality attribute (CQA) that requires careful control and monitoring during the drug discovery and production processes. The unwanted formation of isoAsp within biotherapeutics and resultant structural changes in the peptide backbone may negatively impact the efficacy, potency, and safety of the molecule or become immunogenic, especially if the isomerization occurs within the mAb complementarity determining region (CDR). Herein we describe a MALDI-TOF/TOF mass spectrometry method that affords unequivocal identification of the presence and the exact position of the isoAsp residue(s) in peptide standards ranging in size from a tripeptide to a docosapeptide (22 residues). In general, the peptide bond immediately N-terminal to the isoAsp residue is more susceptible to MALDI-TOF/TOF fragmentation than its unmodified counterpart. In some of the peptides evaluated in this study, fragmentation of the peptide bond C-terminal to the isoAsp residue (the aspartate effect) is also enhanced when compared to the control. Relative quantification by MALDI-TOF/TOF of this chemical modification is dependent upon a successful reversed-phase HPLC (rpHPLC) separation of the control and modified peptides. This method has also been validated on a therapeutic mAb that contains a well-documented isoAsp residue in the heavy chain CDR3 after forced degradation. Moreover, we also demonstrate that higher energy C-trap dissociation of only the singly charged species, and not the multiply charged form, of the isoAsp containing peptide, separated by rpHPLC, results in LC-MS/MS fragmentation that is highly consistent to that of MALDI-TOF/TOF.

Targeting Endogenous Adduction Level of Serum Albumin by Parallel Reaction Monitoring via Standard Additions and Intact Protein Measurement: Biological Dosimetry of Catechol Estrogens.

Abundant blood proteins adducted by active electrophiles are excellent markers to predict the risk of electrophile-induced toxicity. However, detecting endogenously adducted proteins by bottom-up selective (or parallel) reaction monitoring (SRM/PRM) is challenging because of the high variability in sample preparation and detection as well as low adduction levels. Here, we reported a new approach in developing PRM methods by combining intact protein measurement with standard additions to target optimal conditions for detecting catechol estrogens (CEs)-adducted human serum albumin (HSA). Blood serum was added with multiple amounts of CEs to obtain serum standards. Intact protein measurement revealed two linear ranges of adduction levels (adducted-CE/HSA): 0.34-0.42 (R2 > 0.94) and 0.81-8.54 (R2 > 0.96) against the amount of added CEs, respectively. Six adduction sites were identified by trypsin (K20, C34, K73, K281, H338, K378) or chymotrypsin (K20, C34, K378) digestion. PRM methods targeting all adducted/nonadducted peptide pairs based on chymotrypsin or trypsin digestion were developed, and the data were compared with those obtained by intact protein measurement. Correlation plots indicated that chymotrypsin-PRM leads to poor sensitivity and largely underestimated protein adduction levels. Trypsin-PRM leads to sensitive and highly correlated (R2 > 0.91) protein adduction levels with a detection limit below the endogenous level and relative standard deviation <25%. As a proof of concept, clinical serum samples were examined by trypsin-PRM, and a slightly higher adduction level was observed for the obesity group when compared with the healthy group. This is the first report on determining adduction levels of blood proteins for long-term exposure to CEs. The standard addition approach can be generally applied to protein adductomics with resolvable mass increments by intact protein measurement to accelerate the development of bottom-up methods close to the inherent limit.

A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.

TKO6: A Peptide Standard To Assess Interference for Unit-Resolved Isobaric Labeling Platforms.

Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses.

Rapidly Assessing the Quality of Targeted Proteomics Experiments through Monitoring Stable-Isotope Labeled Standards.

Targeted proteomics experiments based on selected reaction monitoring (SRM) have gained wide adoption in the use of clinical biomarkers, cellular modeling, and numerous other biological experiments due to their highly accurate and reproducible quantification. The quantitative accuracy in targeted proteomics experiments is reliant on the stable-isotope, heavy-labeled peptide standards that are spiked into a sample and used as a reference when calculating the abundance of endogenous peptides. Therefore, the quality of measurement for these standards is a critical factor in determining whether data acquisition was successful. With improved mass spectrometry (MS) instrumentation that enables the monitoring of hundreds of peptides in hundreds to thousands of samples, quality assessment is increasingly important and cannot be performed manually. We present Q4SRM, a software tool that rapidly checks the signal from all heavy-labeled peptides and flags those that fail quality-control metrics. Using four metrics, the tool detects problems with both individual SRM transitions and the collective group of transitions that monitor a single peptide. The program's speed and simplicity enable its use at the point of data acquisition and can be ideally run immediately upon the completion of a liquid chromatography-SRM-MS analysis.

Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Quantitative Assessment of the Absolute Purity of Thiopeptcin Reference Standard by 1H-NMR.

Quantitative nuclear magnetic resonance (qNMR) has emerged as an easy, rapid and reproducible method for various pharmaceuticals. In the current study, a general qNMR approach for calibrating the purity of the thiopeptcin reference standard (also known as nocathiacin I) was developed using sulfadoxine as an internal standard. Experimental conditions, such as the relaxation delay time and number of scans, were systematically optimized, and the method was validated with different analytical parameters, including selectivity, stability, linearity, precision and robustness. To examine the reliability and feasibility of the present qNMR method, there was no significant difference in the quantification of this complex cyclic peptide compared to the mass balance method. The present study further exemplified that qNMR is a reliable and valuable approach for the assessing of absolute purity of small-molecule pharmaceuticals, which provides a useful tool for drug discovery and development.

Assessing MS-based quantitation strategies for low-level impurities in peptide reference materials: application to angiotensin II.

Identification and quantitation of related impurities is vital in obtaining corrected purity values for peptide certified reference materials. The sensitivity and selectivity of high-resolution mass spectrometry (MS) renders it an indispensable technique in this arena. Typical quantitation efforts involve constructing external calibration curves, although analysis of dilute peptide solutions can be complicated by analyte adsorption to vial walls, instrument tubing, etc. The standard addition method alleviates many concerns associated with this sample loss as the calibrant solutions more closely match the matrix of the samples. Yet, both strategies require acquisition of synthetic impurity peptide standards. Label-free proteomics relies on electrospray ionization (ESI)-MS signals to quantify identical peptides across multiple samples; however, peptides of differing sequence can exhibit widely disparate ESI-MS responses. This study explores the use of peak area ratios to quantitate sequence-related peptide impurities in an angiotensin II candidate certified reference material. Using synthetic standards of five abundant substances, impurity mass fractions calculated via the relative response method are in reasonable agreement with those determined from standard addition experiments, whereas external calibration measurements frequently overestimate impurity amounts. For a synthetic peptide and its related sequence impurities, the relative response method can expedite analysis and lower expenditures, and in some cases improve data quality.

Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II.

The purity value assignment of metrologically traceable peptide reference standards requires specialized primary methods. Conventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides, thus eliminating potential bias due to hydrolysis. Both methods are susceptible to interference from related peptide impurities, which need to be accurately measured and accounted for. The mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified. Ideally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards. Here we report a novel strategy for correcting both LC-MS/MS and 1H-qNMR results for related peptide impurities and combining results from both methods using a Bayesian statistical approach using mass balance results as prior knowledge. The mass balance method relied on a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass. Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods. The final purity value assignment of the candidate CRM was 691 ± 9 mg/g (k = 2).

Current treatment paradigm and landscape for the management of chronic idiopathic constipation in adults: Focus on plecanatide.

BACKGROUND AND PURPOSE: Chronic idiopathic constipation (CIC) is a prevalent disorder affecting productivity, quality of life, and health care resource utilization. Nurse practitioners (NPs) play a critical function in managing patients presenting with CIC, with roles including evaluation, diagnosis, treatment decisions, and patient education. For adults with inadequate response or tolerability issues using over-the-counter treatments, three prescription agents (plecanatide, linaclotide, and lubiprostone) are available in the United States to treat CIC, of which plecanatide was mostly recently approved. This review provides NPs with a current overview and summary of plecanatide in the current treatment landscape for CIC. METHODS: PubMed was searched for the literature regarding clinical practice guidelines and published trial data for lubiprostone, linaclotide, and plecanatide in CIC. CONCLUSIONS: Efficacy and safety comparisons between prescription agents are limited beacause of the differences in trial duration and primary end points (all different). Generally, plecanatide and linaclotide demonstrated similar efficacy, with plecanatide demonstrating lower rates of adverse events. IMPLICATIONS FOR PRACTICE: The success of CIC treatment can be affected by patient adherence to the regimen, which is dependent on the efficacy and tolerability of treatment. Plecanatide is a promising option for patients whose CIC symptoms are not adequately controlled using their current treatment approach.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS.

An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999), sensitivity (limit of quantitation, 0.16 mg/100 g), recovery (83.1-91.6%), precision (RSD < 5.4%) and repeatability (RSD < 7.7%). To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60-150.56 mg/100 g.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

Evaluating the Characteristics of Reporter Ion Signal Acquired in the Orbitrap Analyzer for Isobaric Mass Tag Proteome Quantification Experiments.

Multiplexed quantification with isobaric chemical tags (e.g., TMT, iTRAQ) provides a robust and efficient means to comparatively examine proteome dynamics between several biological states using a mass spectrometer (MS). The quantitative nature of isobaric tags necessitates strict validation of the observed ion signals in the chosen MS detector before differential patterns are extracted between biological states. We present an in-depth analysis of isobaric tag data acquired on current generation Orbitrap MS hardware to illustrate pitfalls in acquisition settings that can negatively impact results. We establish, for the first time, the presence of a notch, a region of no observed values, in the reporter ion distributions from isobaric-labeled peptide mixtures acquired on these instruments. We determine that this notch is present in published data across a wide range of instruments of the same or different type and is isolated to the Orbitrap mass analyzer. We demonstrate that the impact of the notch can be minimized using manipulations of Orbitrap scan parameters and on-column injection amounts. Lastly, using a mixture of synthetic standard peptides we investigated the impact on identification rates and quantification precision. Together, these data highlight an important phenomenon that negatively impacts peptide identification and quantification in the Orbitrap analyzer as well as outlining guidelines to follow to ensure minimization of MS-induced artifacts in isobaric tag experiments resulting from the notch.

Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry.

Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine.

Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-L-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman's reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.

Recommended administered activities for (68)Ga-labelled peptides in paediatric nuclear medicine.

PURPOSE: The aim of this study was to establish a method for determining administered activities for (68)Ga-labelled peptides. Dose calculations were based on the weight-independent effective dose model proposed by the EANM paediatric dosage card for use in paediatric nuclear medicine. METHODS: Previously published time-integrated activity coefficients for (68)Ga-DOTATATE, (68)Ga-DOTATOC and (68)Ga-pentixafor were used to calculate age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card to determine the radiopharmaceutical class of  (68)Ga-labelled peptides ("multiples") and to calculate the baseline activities based on an upper limit for administered activity (185 MBq) in an adult. RESULTS: All calculated normalization factors suggest that the (68)Ga-labelled peptides are class "B" radiopharmaceuticals. The baseline activity for all compounds is 12.8 MBq. In analogy to (18)F-fluoride, we recommend a minimum activity of 14 MBq. CONCLUSION: For paediatric nuclear medicine applications involving (68)Ga-labelled peptides, we suggest determining administered activities based on the formalism proposed in this work. The corresponding effective doses from these procedures will remain age-independent.

Operation resistance: A snapshot of falsified antibiotics and biopharmaceutical injectables in Europe.

Operation Pangea is an annual international week of action combating pharmaceutical crime. In this study, called Operation Resistance, we asked the national agencies in Europe to search for falsified antibiotics and biopharmaceutical injectables (peptides and proteins) amongst the medicines seized in Pangea 7 (2014). Reports were received from Belgium, Cyprus, Czech Republic, Denmark, France, the Netherlands, Portugal, Sweden, Spain, the United Kingdom, Norway, and Switzerland. The countries reported seizing about 21,000 dose units (e.g. tablets, capsules) of falsified antibiotics in total. Most of the antibiotics were unlicensed medicines with common antibiotic drugs. In this study week, very few falsified biopharmaceutical injectables were reported. Laboratories reported human growth hormone, sermorelin, melanotan II, and no active ingredients. The average shipment size seemed too large for personal use indicating that a substantial part was intended for resale. This study provides a snapshot of the falsified antibiotics and biopharmaceuticals that enter European countries. How much is actually reaching users during Pangea week - in on other weeks - remains unknown. The shipment sizes indicate falsified antibiotics and biopharmaceuticals are imported for both personal use and resale. The use of antibiotics from unreliable sources is a health risk, contributes to antimicrobial resistance, and may obscure a source of infection from health agencies. The falsified biopharmaceuticals are a health risk because they lack all labelling and may contain unlicensed drugs for injection. It seems important to raise awareness among health-care professionals that falsified medicines in Europe are not restricted to erectile dysfunction drugs. Copyright © 2015 John Wiley & Sons, Ltd.