Daniel G. Delafield

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity (sn-positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic N,N-dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL sn-isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled sn-isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL sn-isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of sn-isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL sn-isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL sn-isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.

Dysregulated glycerophospholipid (GP) metabolism in the brain is associated with the progression of neurodegenerative diseases including Alzheimer’s disease (AD). Routine liquid chromatography-mass spectrometry (LC-MS)-based large-scale lipidomic methods often fail to elucidate subtle yet important structural features such as sn-position, hindering the precise interrogation of GP molecules. Leveraging high-resolution demultiplexing (HRdm) ion mobility spectrometry (IMS), we develop a four-dimensional (4D) lipidomic strategy to resolve GP sn-position isomers. We further construct a comprehensive experimental 4D GP database of 498 GPs identified from the mouse brain and an in-depth extended 4D library of 2500 GPs predicted by machine learning, enabling automated profiling of GPs with detailed acyl chain sn-position assignment. Analyzing three mouse brain regions (hippocampus, cerebellum, and cortex), we successfully identify a total of 592 GPs including 130 pairs of sn-position isomers. Further temporal GPs analysis in the three functional brain regions illustrates their metabolic alterations in AD progression.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1^KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.

The ubiquity of mass spectrometry-based bottom-up proteomic analyses as a component of biological investigation mandates the validation of methodologies that increase acquisition efficiency, improve sample coverage, and enhance profiling depth. Chromatographic separation is often ignored as an area of potential improvement, with most analyses relying on traditional reversed-phase liquid chromatography (RPLC); this consistent reliance on a single chromatographic paradigm fundamentally limits our view of the observable proteome. Herein, we build upon early reports and validate porous graphitic carbon chromatography (PGC) as a facile means to substantially enhance proteomic coverage without changes to sample preparation, instrument configuration, or acquisition methods. Analysis of offline fractionated cell line digests using both separations revealed an increase in peptide and protein identifications by 43% and 24%, respectively. Increased identifications provided more comprehensive coverage of cellular components and biological processes independent of protein abundance, highlighting the substantial quantity of proteomic information that may go undetected in standard analyses. We further utilize these data to reveal that label-free quantitative analyses using RPLC separations alone may not be reflective of actual protein constituency. Together, these data highlight the value and comprehension offered through PGC-MS proteomic analyses. RAW proteomic data have been uploaded to the MassIVE repository with the primary accession code MSV000091495.

Analytical and technological advancements in multidimensional ion mobility-mass spectrometry and molecular dynamic simulations catalyze the birth of a new precise strategy for fine-tuning Aβ42 structure and cytotoxicity.

Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes, and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomic experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomic and glycoproteomic datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively. Graphical abstract

Citrullination and homocitrullination are key post-translational modifications (PTMs) that affect protein structures and functions. Although they have been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize these PTMs. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of these two PTMs simultaneously via mass spectrometry. We perform global mapping of the citrullination and homocitrullination proteomes of mouse tissues. In total, we identify 691 citrullination sites and 81 homocitrullination sites from 432 and 63 proteins, respectively, representing the largest datasets to date. We discover novel distribution and functions of these two PTMs. We also perform multiplexing quantitative analysis via isotopic labeling techniques. This study depicts a landscape of protein citrullination and homocitrullination and lays the foundation for further deciphering their physiological and pathological roles.