Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

Dynamic Data-Independent Acquisition Mass Spectrometry with Real-Time Retrospective Alignment.

Data-independent acquisition (DIA) mass spectrometry has grown in popularity in recent years, because of the reproducibility and quantitative rigor of a systematic tandem mass spectrometry (MS/MS) sampling method. However, traditional DIA methods may spend valuable instrument time acquiring MS/MS spectra with no usable information in them, affecting sensitivity and quantitative performance. We developed a DIA strategy that dynamically adjusts the MS/MS windows during the chromatographic separation. The method focuses MS/MS acquisition on the most relevant mass range at each point in time─increasing the quantitative sensitivity by increasing the time spent on each DIA window. We demonstrate an improved lower limit of quantification, on average, without sacrificing the number of peptides detected.

Putting Humpty Dumpty Back Together Again: What Does Protein Quantification Mean in Bottom-Up Proteomics?

Bottom-up proteomics provides peptide measurements and has been invaluable for moving proteomics into large-scale analyses. Commonly, a single quantitative value is reported for each protein-coding gene by aggregating peptide quantities into protein groups following protein inference or parsimony. However, given the complexity of both RNA splicing and post-translational protein modification, it is overly simplistic to assume that all peptides that map to a singular protein-coding gene will demonstrate the same quantitative response. By assuming that all peptides from a protein-coding sequence are representative of the same protein, we may miss the discovery of important biological differences. To capture the contributions of existing proteoforms, we need to reconsider the practice of aggregating protein values to a single quantity per protein-coding gene.

VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis.

Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

Multiplexed MS/MS for improved data-independent acquisition.

In mass spectrometry-based proteomics, data-independent acquisition (DIA) strategies can acquire a single data set useful for both identification and quantification of detectable peptides in a complex mixture. However, DIA data are noisy owing to a typical five- to tenfold reduction in precursor selectivity compared to data obtained with data-dependent acquisition or selected reaction monitoring. We demonstrate a multiplexing strategy, MSX, for DIA analysis that increases precursor selectivity fivefold.

Maximizing peptide identification events in proteomic workflows using data-dependent acquisition (DDA).

Current analytical strategies for collecting proteomic data using data-dependent acquisition (DDA) are limited by the low analytical reproducibility of the method. Proteomic discovery efforts that exploit the benefits of DDA, such as providing peptide sequence information, but that enable improved analytical reproducibility, represent an ideal scenario for maximizing measureable peptide identifications in "shotgun"-type proteomic studies. Therefore, we propose an analytical workflow combining DDA with retention time aligned extracted ion chromatogram (XIC) areas obtained from high mass accuracy MS1 data acquired in parallel. We applied this workflow to the analyses of sample matrixes prepared from mouse blood plasma and brain tissues and observed increases in peptide detection of up to 30.5% due to the comparison of peptide MS1 XIC areas following retention time alignment of co-identified peptides. Furthermore, we show that the approach is quantitative using peptide standards diluted into a complex matrix. These data revealed that peptide MS1 XIC areas provide linear response of over three orders of magnitude down to low femtomole (fmol) levels. These findings argue that augmenting "shotgun" proteomic workflows with retention time alignment of peptide identifications and comparative analyses of corresponding peptide MS1 XIC areas improve the analytical performance of global proteomic discovery methods using DDA.

Mass spectrometry accelerates membrane protein analysis.

Cellular membranes are composed of proteins and glyco- and phospholipids and play an indispensible role in maintaining cellular integrity and homeostasis, by physically restricting biochemical processes within cells and providing protection. Membrane proteins perform many essential functions, which include operating as transporters, adhesion-anchors, receptors, and enzymes. Recent advancements in proteomic mass spectrometry have resulted in substantial progress towards the determination of the plasma membrane (PM) proteome, resolution of membrane protein topology, establishment of numerous receptor protein complexes, identification of ligand-receptor pairs, and the elucidation of signaling networks originating at the PM. Here, we discuss the recent accelerated success of discovery-based proteomic pipelines for the establishment of a complete membrane proteome.

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer.

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the Stellar™ mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

A framework for quality control in quantitative proteomics.

A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow from planning to analysis. We share real-world case studies applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at protein and peptide-level allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis using Skyline, longitudinal QC metrics using AutoQC, and server-based data deposition using PanoramaWeb. We propose that this integrated approach to QC be used as a starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible.

Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome.

Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent ~1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 µL plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimer's disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinson's disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 ± 0.06.

Improved precision of proteomic measurements in immunoprecipitation based purifications using relative quantitation.

Mass spectrometry coupled immunoprecipitation (MS-IP) studies are useful in identifying and quantitating potential binding partners of a target protein. However, they are often conducted without appropriate loading controls. Western blots are often used to analyze loading controls, yet there are limitations to their usefulness as analytical tools. One remedy for this is the use of selected reaction monitoring (SRM), where the areas under the curve (AUCs) of peptides from a protein of interest can be normalized to those from the constant regions of the immunoglobulins used for the IP. Using this normalization method, significant changes in relative peptide abundance were observed between samples when there appeared to be an unequal load based on immunoglobulin peptide abundance.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific™ Orbitrap™ mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis.

Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.

Tandem Mass Spectrometry-Based Amyloid Typing Using Manual Microdissection and Open-Source Data Processing.

OBJECTIVES: Standard implementations of amyloid typing by liquid chromatography-tandem mass spectrometry use capabilities unavailable to most clinical laboratories. To improve accessibility of this testing, we explored easier approaches to tissue sampling and data processing. METHODS: We validated a typing method using manual sampling in place of laser microdissection, pairing the technique with a semiquantitative measure of sampling adequacy. In addition, we created an open-source data processing workflow (Crux Pipeline) for clinical users. RESULTS: Cases of amyloidosis spanning the major types were distinguishable with 100% specificity using measurements of individual amyloidogenic proteins or in combination with the ratio of λ and κ constant regions. Crux Pipeline allowed for rapid, batched data processing, integrating the steps of peptide identification, statistical confidence estimation, and label-free protein quantification. CONCLUSIONS: Accurate mass spectrometry-based amyloid typing is possible without laser microdissection. To facilitate entry into solid tissue proteomics, newcomers can leverage manual sampling approaches in combination with Crux Pipeline and related tools.

Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling.

BACKGROUND: Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins. RESULTS: We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract. CONCLUSION: Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.

Proteomic insights into an expanded cellular role for cytoplasmic lipid droplets.

Cytoplasmic lipid droplets (CLDs) are cellular structures composed of a neutral lipid core surrounded by a phospholipid monolayer of amphipathic lipids and a variety of proteins. CLDs have classically been regarded as cellular energy storage structures. However, recent proteomic studies reveal that, although many of the proteins found to associate with CLDs are connected to lipid metabolism, storage, and homeostasis, there are also proteins with no obvious connection to the classical function and typically associated with other cellular compartments. Such proteins are termed refugee proteins, and their presence suggests that CLDs may serve an expanded role as a dynamic protein storage site, providing a novel mechanism for the regulation of protein function and transport.

Quantitative improvements in peptide recovery at elevated chromatographic temperatures from microcapillary liquid chromatography-mass spectrometry analyses of brain using selected reaction monitoring.

Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using selected reaction monitoring (SRM) mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in mouse whole brain matrix are highlighted, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in microcapillary liquid chromatography-mass spectrometry (muLC-MS) based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.

Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics.

The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput. When merged with targeted mass spectrometry, this pipeline can fuel hypothesis-driven studies and the development of novel diagnostics and therapeutics.