Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, we outline some recent advances and discuss current challenges and future directions for the field

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics

Intact Protein Analysis at 21 Tesla and X-Ray Crystallography Define Structural Differences in Single Amino Acid Variants of Human Mitochondrial Branched-Chain Amino Acid Aminotransferase 2 (BCAT2)

Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.]

Discovery of a biomarker for β-Thalassemia by HPLC-MS and improvement from Proton Transfer Reaction – Parallel Ion Parking

β-thalassemia is a quantitative hemoglobin (Hb) disorder resulting in reduced production of Hb A and increased levels of Hb A2. Diagnosis of β-thalassemia can be problematic when combined with other structural Hb variants, so that the separation approaches in routine clinical centers are not sufficiently decisive to obtain accurate results. Here, we separate the intact Hb subunits by high-performance liquid chromatography, followed by top-down tandem mass spectrometry of intact subunits to distinguish Hb variants. Proton transfer reaction-parallel ion parking (PTR-PIP), in which a radical anion removes protons from multiply charged precursor ions and produces charge-reduced ions spanning a limited m/z range, was used to increase the signal-to-noise ratio of the subunits of interest. We demonstrate that the δ/β ratio can act as a biomarker to identify β-thalassemia in normal electrospray ionization MS1 and PTR-PIP MS1. The application of PTR-PIP significantly increases the sensitivity and specificity of the HPLC-MS method to identify δ/β ratio as a thalassemia biomarker

Construction of Human Proteoform Families from 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Top-Down Proteomic Data

Identification of proteoforms, the different forms of a protein, is important to understand biological processes. A proteoform family is the set of different proteoforms from the same gene. We previously developed the software program Proteoform Suite, which constructs proteoform families and identifies proteoforms by intact-mass analysis. Here, we have applied this approach to top-down proteomic data acquired at the National High Magnetic Field Laboratory 21 tesla Fourier transform ion cyclotron resonance mass spectrometer (data available on the MassIVE platform with identifier MSV000085978). We explored the ability to construct proteoform families and identify proteoforms from the high mass accuracy data that this instrument provides for a complex cell lysate sample from the MCF-7 human breast cancer cell line. There were 2830 observed experimental proteforms, of which 932 were identified, 44 were ambiguous, and 1854 were unidentified. Of the 932 unique identified proteoforms, 766 were identified by top-down MS2 analysis at 1% false discovery rate (FDR) using TDPortal, and 166 were additional intact-mass identifications (∼4.7% calculated global FDR) made using Proteoform Suite. We recently published a proteoform level schema to represent ambiguity in proteoform identifications. We implemented this proteoform level classification in Proteoform Suite for intact-mass identifications, which enables users to determine the ambiguity levels and sources of ambiguity for each intact-mass proteoform identification

Advanced Strategies for Proton-Transfer Reactions Coupled with Parallel Ion Parking on a 21 T FT-ICR MS for Intact Protein Analysis

Proton-transfer reactions (PTRs) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to "concentrate", in the gas phase, nearly all the signals from an intact protein charge state envelope into a single charge state, improving the signal-to-noise ratio (S/N) by 10× or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides a tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call "PTR-MS1-PIP", generates a pseudo-MS1 spectrum derived from ions that are exposed to the PTR reagent and PIP waveforms but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in the spectral S/N of intact proteins. This permits the observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the PTR rate without compromise to the parking efficiency. We demonstrate that this process, called "rapid park", can improve reaction rates by 5-10× and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS1 and rapid park provides a 10-fold reduction in ion injection time, improving the rate of tandem MS sequencing

The developmental order of emotional and cognitive empathy in adolescents, and the role of mothers

This four-year study with annual measurements investigated the longitudinal interplay between affective and cognitive empathy in adolescents and their mothers. We studied 1) whether the developmental order of empathy in adolescence progresses from affective to cognitive empathy, or vice versa; 2) whether mothers’ empathy predicts their children’s empathy development; 3) whether adolescent gender moderates such intergenerational transmission from mothers to adolescents; and 4) whether inter-respondent differences in empathy were more stable for affective or cognitive empathy, and for adolescents or for mothers. Results indicated that affective empathy positively predicted the development of cognitive empathy one year later, but not vice versa. Mothers’ greater cognitive empathy predicted increasing cognitive empathy over time in daughters, but not in sons. Inter-respondent differences were more stable for affective empathy than for cognitive empathy in adolescents. In mothers, both empathy dimensions were equally stable, and more stable than in adolescents. This study thereby suggests that the developmental order of empathy in adolescence progresses from affective to cognitive empathy, in contrast to prior experimental and theoretical work which has emphasized the reverse direction of effects. It further offers support for the intergenerational transmission of cognitive empathy over time. Together with the lower stability of cognitive empathy, these findings suggest that adolescence is a developmentally sensitive period for cognitive empathy