MS-Based Proteomics of Body Fluids: The End of the Beginning.

Accurate biomarkers are a crucial and necessary precondition for precision medicine, yet existing ones are often unspecific and new ones have been very slow to enter the clinic. Mass spectrometry (MS)-based proteomics excels by its untargeted nature, specificity of identification, and quantification, making it an ideal technology for biomarker discovery and routine measurement. It has unique attributes compared to affinity binder technologies, such as OLINK Proximity Extension Assay and SOMAscan. In in a previous review in 2017, we described technological and conceptual limitations that had held back success. We proposed a 'rectangular strategy' to better separate true biomarkers by minimizing cohort-specific effects. Today, this has converged with advances in MS-based proteomics technology, such as increased sample throughput, depth of identification, and quantification. As a result, biomarker discovery studies have become more successful, producing biomarker candidates that withstand independent verification and, in some cases, already outperform state-of-the-art clinical assays. We summarize developments over the last years, including the benefits of large and independent cohorts, which are necessary for clinical acceptance. Shorter gradients, new scan modes, and multiplexing are about to drastically increase throughput, cross-study integration, and quantification, including proxies for absolute levels. We have found that multiprotein panels are inherently more robust than current single analyte tests and better capture the complexity of human phenotypes. Routine MS measurement in the clinic is fast becoming a viable option. The full set of proteins in a body fluid (global proteome) is the most important reference and the best process control. Additionally, it increasingly has all the information that could be obtained from targeted analysis although the latter may be the most straightforward way to enter regular use. Many challenges remain, not least of a regulatory and ethical nature, but the outlook for MS-based clinical applications has never been brighter.

Tricalbin proteins regulate plasma membrane phospholipid homeostasis.

The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain incomplete. Among the E-Syt family, the yeast tricalbin (Tcb) proteins are essential for PM integrity upon heat stress, but it is not known how they contribute to PM maintenance. Using quantitative lipidomics and microscopy, we find that the Tcb proteins regulate phosphatidylserine homeostasis at the PM. Moreover, upon heat-induced membrane stress, Tcb3 co-localises with the PM protein Sfk1 that is implicated in PM phospholipid asymmetry and integrity. The Tcb proteins also control the PM targeting of the known phosphatidylserine effector Pkc1 upon heat-induced stress. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We propose that phospholipid regulation is an ancient essential function of E-Syt family members required for PM integrity.

Cell-Type- and Brain-Region-Resolved Mouse Brain Lipidome.

Gene and protein expression data provide useful resources for understanding brain function, but little is known about the lipid composition of the brain. Here, we perform quantitative shotgun lipidomics, which enables a cell-type-resolved assessment of the mouse brain lipid composition. We quantify around 700 lipid species and evaluate lipid features including fatty acyl chain length, hydroxylation, and number of acyl chain double bonds, thereby identifying cell-type- and brain-region-specific lipid profiles in adult mice, as well as in aged mice, in apolipoprotein-E-deficient mice, in a model of Alzheimer's disease, and in mice fed different diets. We also integrate lipid with protein expression profiles to predict lipid pathways enriched in specific cell types, such as fatty acid ß-oxidation in astrocytes and sphingolipid metabolism in microglia. This resource complements existing brain atlases of gene and protein expression and may be useful for understanding the role of lipids in brain function.

Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease.

Neurodegenerative diseases are a growing burden, and there is an urgent need for better biomarkers for diagnosis, prognosis, and treatment efficacy. Structural and functional brain alterations are reflected in the protein composition of cerebrospinal fluid (CSF). Alzheimer's disease (AD) patients have higher CSF levels of tau, but we lack knowledge of systems-wide changes of CSF protein levels that accompany AD. Here, we present a highly reproducible mass spectrometry (MS)-based proteomics workflow for the in-depth analysis of CSF from minimal sample amounts. From three independent studies (197 individuals), we characterize differences in proteins by AD status (> 1,000 proteins, CV < 20%). Proteins with previous links to neurodegeneration such as tau, SOD1, and PARK7 differed most strongly by AD status, providing strong positive controls for our approach. CSF proteome changes in Alzheimer's disease prove to be widespread and often correlated with tau concentrations. Our unbiased screen also reveals a consistent glycolytic signature across our cohorts and a recent study. Machine learning suggests clinical utility of this proteomic signature.

The proteome landscape of the kingdoms of life.

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported1, advances in mass-spectrometry-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3-6. However, there have been few comparisons across species7,8, in stark contrast with genomics initiatives9. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.

A heat-sensitive Osh protein controls PI4P polarity.

BACKGROUND: Phosphoinositide lipids provide spatial landmarks during polarized cell growth and migration. Yet how phosphoinositide gradients are oriented in response to extracellular cues and environmental conditions is not well understood. Here, we elucidate an unexpected mode of phosphatidylinositol 4-phosphate (PI4P) regulation in the control of polarized secretion. RESULTS: We show that PI4P is highly enriched at the plasma membrane of growing daughter cells in budding yeast where polarized secretion occurs. However, upon heat stress conditions that redirect secretory traffic, PI4P rapidly increases at the plasma membrane in mother cells resulting in a more uniform PI4P distribution. Precise control of PI4P distribution is mediated through the Osh (oxysterol-binding protein homology) proteins that bind and present PI4P to a phosphoinositide phosphatase. Interestingly, Osh3 undergoes a phase transition upon heat stress conditions, resulting in intracellular aggregates and reduced cortical localization. Both the Osh3 GOLD and ORD domains are sufficient to form heat stress-induced aggregates, indicating that Osh3 is highly tuned to heat stress conditions. Upon loss of Osh3 function, the polarized distribution of both PI4P and the exocyst component Exo70 are impaired. Thus, an intrinsically heat stress-sensitive PI4P regulatory protein controls the spatial distribution of phosphoinositide lipid metabolism to direct secretory trafficking as needed. CONCLUSIONS: Our results suggest that control of PI4P metabolism by Osh proteins is a key determinant in the control of polarized growth and secretion.

Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies.

Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)-based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike-in experiments, we determine sample quality-associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We provide sample preparation guidelines and an online resource ( www.plasmaproteomeprofiling.org) to assess overall sample-related bias in clinical studies and to prevent costly miss-assignment of biomarker candidates.

Phosphoinositide kinase signaling controls ER-PM cross-talk.

Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase-mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions.