It's a bumper pod! David Price leads Ashleigh Macro and Henry Burrell down the topical rabbit hole to discuss why Black Friday largely sucks, but is an interesting venture for publishers as well as consumers. Who else bought a Switch?

We then tackle the Mac root issue that hit headlines worldwide before tearing the Pixel Buds a new one. And we all downloaded Animal Crossing: Pocket Camp to see what the fuss is about.

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We finally get the IoT acronym the pod has been waiting for as we discuss Bitcoin and 17th century Dutch finance. Charlotte Jee informs Henry Burrell, David Price and Scott Carey.

Scott then leads us down the winding road of the so-called platform wars, in a week when Google blocked the Amazon Echo Show from streaming YouTube. Is this a bump in the road or will services be cut from rival hardware?

And finally, led by David, the pod discusses its favourite tech of the year and what you might consider buying for friends and family for Christmas.

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Once more for 2017 as Henry Burrell, Karen Khan and Scott Carey bid farewell to this wonderful year (ahem) with musings on Apple's sexy new iMac Pro. Who is it for, how much is it and does this mean there is no Mac Pro in 2018?

We then tackle Netflix's Twitter shaming of its users and why Spotify got away with it earlier in the year. How comfortable are we all when we realise how much data companies really have on us?

In light of this, Apple bought Shazam - most likely for the data sets as much as the tech and the talent. What form will it take in Apple as another UK tech company is acquired?

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2018 lands with a distinct thud as Charlotte Jee tackles Meltdown and Spectre, David Price wrestles with Apple's batteries and its new iMac Pro, before Miriam Harris works through the Bafta nominations for visual effects. Henry Burrell leads us down the rabbit hole.

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This week host Charlotte Jee breaks down open banking with Computerworld UK editor Scott Carey: what is it and why should we care?

Then audience development editor Christina Mercer explains why chip-maker Qualcomm has been fined nearly €1 billion and the EU's sustained attack on big tech (12:00)

Last up is senior staff writer at Tech Advisor Dom Preston to talk about Amazon's revolutionary concept Go store opening in Seattle and if this is really the future of shopping (20:00).

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A double Google header as Henry Burrell leads Scott Carey and Sean Bradley down the tech rabbit hole. Alphabet's most famous outlet has redesigned Gmail for desktop, but what does that mean for businesses and consumers. Will anyone notice?

Then what is Chat? Not Google Chat or Android Chat, mind - just Chat. Will the Google-led initiative really transform text messaging on Android or is it doomed to forever to be a fragmented mess?

Sean then talks us through the latest AMD processors to challenge Intel. Intel hasn't had the best of year's after Apple announced it is going solo. Is there a change afoot?

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Oh boy what a meaty session we have for you as Christina Mercer, Somrata Sarkar, David Price and Henry Burrell tackle three whopics (whopping topics) head on.

Somrata takes us into the sometimes scary thoughts of our own digital afterlives. Should we be worried that we'll end up as misrepresentative chat bots one day? Who will have the authority to police the companies that harvest our data?

Then Christina explains the knife edge America is on when it comes to net neutrality. Despite recent hope, there's still a chance the web across the pond will be ruthlessly metered and segmented.

Finally David asks us if we've checked our unused email accounts recently, as there might be a lot of desperate noodle companies in there begging you to stay on their mailing lists.

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Join host Henry Burrell in hot as hell London town to bring you 40 minutes of air conditioned tech chat.

Tamlin Magee talks us through the murky ways IBM helped the Third Reich in the Thirties and Forties with data collection and asks what responsibility tech companies have today to ensure their work does not contribute to evil.

Charlotte Jee then analyses Facebook's stock price slump, asking why it happened and does it really affect the company? The team muses on Facebook as a whole and the fascinating if polarising Zuckerberg.

Finally Macworld's David Price chats about the new MacBook Pros and how Apple has already fixed the major flaw in the high-end model - but why did they ship this way? Is Apple less concerned with quality control these days?

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This week our host Scott Carey is joined by Macworld UK editor Karen Khan to chat about Apple's latest blockbuster results.

Then group production editor Tamlin Magee jumps in to discuss Amazon's working practices following the collective action around Prime Day.

Finally, Scott chats through his experience at the Google Cloud Next conference in San Francisco last week to see how it is trying to compete with the big boys at Amazon and Microsoft.

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This week our host Scott Carey is joined by Techworld editor Charlotte Jee to discuss the revelation that Google is still tracking users, even if you have that feature disabled, and the wider topic of privacy.

Then games editor at Tech Advisor, Lewis Painter, joins to talk about one of the most hotly anticipated games of the year: Red Dead Redemption 2.

Lastly Macworld UK editor Karen Khan talks about the enduring legacy of the iMac PC 20 years on and how it set Apple on a historic trajectory.

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A quick-fire reaction Apple special, recorded the morning after new iPhones and an Apple Watch were announced at an event in California.

Henry Burrell joins David Price, Ashleigh Macro and Scott Carey to discuss which products we'll be buying, avoiding and coveting over the Christmas period.

Is the new iPhone named terribly? Is it an upgrade at all? What wasn't announced? And does Scott give the new Apple Watch any praise at all? It's all here.

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Two’s a crowd for this week’s games and Christmas pod with Consumer Tech Editor Henry Burrell and Games Editor Lewis Painter. Lewis lines up reviews of the awful Fallout 76 and the excellent online mode of Red Dead Redemption 2 – two polar opposites in how to make a decent online multiplayer game.

We also discuss smartwatches we’ve recently reviewed that left us feeling cold and why Apple is still king of the hill.

And to get into the festive vibe, interspersed throughout as we enter December are our top tech Christmas gift picks, both cheap and indulgent.

Everything we recommend is linked below:

Google Home Hub

Tile Bluetooth tracker

Red Dead Redemption 2 (PS4)

Super Smash Bros – Ultimate (Switch)

Pokémon: Let’s Go, Pikachu! (Switch)

Turtle Beach Elite Pro 2 + Superamp

Amazon Kindle Paperwhite

PlayStation Classic

PlayStation VR Starter Pack

The best budget smartphones

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This week our host Scott Carey catches up with Sean Bradley and Dominic Preston fresh off the back of the International Consumer Electronics Show (CES) in Las Vegas.

Dom explains why he was surprised, both in a good and bad way, by the latest foldable screen technology and Sean talks about what is going on in the gaming laptop space, as Alienware looks to soften its image.

Then Dom talks about a piece of connected wood and the gang break down the big controversy from the show floor regarding a certain innovative dildo.

We are also announcing the sad news that the UK Tech Weekly Podcast will be going on an indefinite hiatus while we reassess our podcasting output. We would like to thank anyone that has taken the time to listen to us for these one hundred and twelve (112!) episodes and rest assured we will be back in some guise soon.

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This manuscript has been withdrawn by the Author.

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98.

Photoreceptors have high energy-demands and a high density of mitochondria that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS) of fuel substrates. Although glucose is the major fuel for central nervous system (CNS) brain neurons, in photoreceptors (also CNS), most glucose is not metabolized through OXPHOS but is instead metabolized into lactate by aerobic glycolysis. The major fuel sources for photoreceptor mitochondria remained unclear for almost six decades. Similar to other tissues (like heart and skeletal muscle) with high metabolic rates, photoreceptors were recently found to metabolize fatty acids (palmitate) through OXPHOS. Disruption of lipid entry into photoreceptors leads to extracellular lipid accumulation, suppressed glucose transporter expression, and a duel lipid/glucose fuel shortage. Modulation of lipid metabolism helps restore photoreceptor function. However, further elucidation of the types of lipids used as retinal energy sources, the metabolic interaction with other fuel pathways, as well as the crosstalk among retinal cells to provide energy to photoreceptors is not yet known. In this review, we will focus on the current understanding of photoreceptor energy demand and sources, and potential future investigations of photoreceptor metabolism.

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism which differs from that of functional inhibitors of ASM (FIASMAs). We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASMpromoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

Nutritional studies rely on various biological specimens for fatty acid (FA) determination, yet it is unclear how levels of serum non-esterified FA (NEFAs) correlate with other circulating lipid pools. Here, we used a high throughput method (< 4 min/sample) based on multisegment injection-non-aqueous-capillary electrophoresis–mass spectrometry (MSI-NACE-MS) to investigate whether specific serum NEFAs have utility as biomarkers of dietary fat intake in women. We first  identified circulating NEFAs correlated with long-term/habitual food intake among pregnant women with contrasting dietary patterns (n=50). Acute changes in serum NEFA trajectories were also studied in non-pregnant women (n=18) following high-dose (5 g/day) fish oil (FO) supplementation or isoenergetic sunflower oil placebo over 56 days. In the cross-sectional study, serum omega-3 (-3) FA correlated with self-reported total -3 daily intake, notably eicosapentaenoic acid (EPA) as its NEFA (r=0.46; p=0.001), whereas pentadecanoic acid was associated with full-fat dairy intake (r=0.43; p=0.002), outcomes consistent with results from  total FA serum hydrolysates. In the intervention cohort, serum -3 NEFAs increased 2.5-fold from baseline within 28 days following FO supplementation, and this increase was most pronounced for EPA (p=0.0004). Unlike for docosahexaenoic acid, circulating EPA as its NEFA also strongly correlated to EPA concentrations measured from erythrocyte phospholipid hydrolysates (r=0.66; p=4.6 x 10-10), and was better suited to detect dietary non-adherence. We conclude that MSI-NACE-MS offers a rapid method to quantify serum NEFAs and objectively monitor dietary fat intake in women that is complementary to diet records or food frequency questionnaires.

Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC–MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from the human and mouse SC. Phytosphingosine-type ceramides (P-ceramides) and 6-hydroxy sphingosine-type ceramides (H-ceramides), which both contain an additional hydroxyl group, were abundant in human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, P-ceramides and H-ceramides were present at ~1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ~90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy FA (B-ceramides) or -hydroxy FA were abundant in mice. The hydroxylated β-carbon in B-ceramides was in the (R)-configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased B-ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the endoplasmic reticulum, is a substrate for B-ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) stratum corneum ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active, epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS. 

Niemann–Pick type C1 (NPC1) disease is a rare genetic condition in which the function of the lysosomal cholesterol transporter NPC1 protein is impaired. Consequently, sphingolipids and cholesterol accumulate in lysosomes of all tissues, triggering a cascade of pathological events that culminate in severe systemic and neurological symptoms. Lysosomal cholesterol accumulation is also a key-factor in the development of atherosclerosis and non-alcoholic steatohepatitis (NASH). In these two metabolic diseases, the administration of plant stanol esters has been shown to ameliorate cellular cholesterol accumulation and inflammation. Given the overlap of pathological mechanisms among atherosclerosis, NASH and NPC1 disease, we sought to investigate whether dietary supplementation with plant stanol esters improves the peripheral features of NPC1 disease. To this end, we used an NPC1 murine model featuring an Npc1 null allele (Npc1^nih), creating a dysfunctional NPC1 protein. Npc1^nih mice were fed a two or six percent plant stanol esters–enriched diet over the course of 5 weeks. During this period, hepatic and blood lipid and inflammatory profiles were assessed. Npc1^nih mice fed the plant stanol–enriched diet exhibited lower hepatic cholesterol accumulation, damage and inflammation than regular chow–fed Npc1^nih mice. Moreover, plant stanol consumption shifted circulating T-cells and monocytes in particular towards an anti-inflammatory profile. Overall, these effects were stronger following dietary supplementation with 6% stanols, suggesting a dose-dependent effect. The findings of our study highlight the potential use of plant stanols as an affordable complementary means to ameliorate disorders in hepatic and blood lipid metabolism and reduce inflammation in NPC1 disease.

Mass spectrometry (MS) assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of optimal cutting temperature compound (OCT) used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT also interferes with protein quantification assays, and that the presence of OCT impacts the quantification of extracted sphingolipids by LC–ESI–MS/MS. We developed and validated a simple and inexpensive method that removes OCT from OCT-embedded tissues. Our results indicate that removal of OCT from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC–ESI–MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer, and to determine the long-term stability of sphingolipids in OCT-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT-cryopreserved normal lung tissues. This validated sphingolipidomic OCT-removal protocol should be a valuable addition to the lipid biologist’s toolbox.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid that promotes lipid sorting in late endosomes/lysosomes by activating lipid hydrolases and lipid transfer proteins. Changes in the cellular BMP content therefore reflect an altered metabolic activity of the endo-lysosomal system. Surprisingly, little is known about the physiological regulation of BMP. In this study, we investigated the effects of nutritional and metabolic factors on BMP profiles of whole tissues and  parenchymal and non-parenchymal cells. Tissue samples were obtained from fed, fasted, two-hours refed, and insulin-treated mice, as well as from mice housed at  5°C, 22°C, or 30°C. These tissues exhibited distinct BMP profiles, which were regulated by the nutritional state in a tissue-specific manner. Insulin treatment was not sufficient to mimic refeeding-induced changes in tissue BMP levels indicating that BMP metabolism is regulated by other hormonal or nutritional factors. Tissue fractionation experiments revealed that fasting drastically elevates BMP levels in hepatocytes and pancreatic cells. Furthermore, we observed that the BMP content in brown adipose tissue strongly depends on housing temperatures. In conclusion, our observations suggest that BMP concentrations adapt to the metabolic state in a tissue-and cell type-specific manner in mice. Drastic changes observed in hepatocytes, pancreatic cells, and brown adipocytes suggest that BMP possesses a role in the functional adaption to nutrient starvation and ambient temperature.

The levels and composition of sphingolipids and related metabolites are altered in aging and common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC–MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified endoplasmic reticulum (ER), mitochondria-associated membranes (MAM), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, sphingomyelin in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine (STS)-induced apoptosis in U251 cells. Ceramide, especially C16-ceramide, levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and sphingomyelin, but sphingosine and lactosyl- and glucosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when sphingomyelin levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase (ASM) activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and ER during the early phases of apoptosis.

Niemann-Pick disease, type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized consensus spectra analysis of MS imaging datasets and orthogonal LC–MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and bisphosphate (PIP2), in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2 α (PI4K2A) in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.

Vitamin A aldehyde covalently bound to opsin protein is embedded in a phospholipid-rich membrane that supports photon absorption and phototransduction in photoreceptor cell outer segments. Following absorption of a photon, the 11-cis-retinal chromophore of visual pigment in photoreceptor cells isomerizes to all-trans-retinal. To maintain photosensitivity 11-cis-retinal must be replaced. At the same time, however, all-trans-retinal has to be handled so as to prevent nonspecific aldehyde activity. Some molecules of retinaldehyde upon release from opsin are efficiently reduced to retinol. Other molecules are released into the lipid phase of the disc membrane where they form a conjugate (N-retinylidene-PE, NRPE) through a Schiff base linkage with phosphatidylethanolamine (PE). The reversible formation of NRPE serves as a transient sink for retinaldehyde that is intended to return retinaldehyde to the visual cycle. However, if instead of hydrolyzing to PE and retinaldehyde, NRPE reacts with a second molecule of retinaldehyde a synthetic pathway is initiated that leads to the formation of multiple species of unwanted bisretinoid fluorophores. We report on recently identified members of the bisretinoid family some of which differ with respect to the acyl chains associated with the glycerol backbone. We discuss processing of the lipid moieties of these fluorophores in lysosomes of retinal pigment epithelial (RPE) cells, their fluorescence characters and new findings related to light and iron-associated oxidation of bisretinoids.

Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems, and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitroendothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivorat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ~73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8h after sepsis induction were lower in rats that survived sepsis than in non-survivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitroexperiments 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.

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Ankylosing Spondylitis (AS) is a common, inflammatory rheumatic disease, which primarily affects the axial skeleton and is associated with sacroiliitis, uveitis and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine if plasma from patients with AS contained autoantibodies and if so, characterize and quantify this response in comparison to patients with Rheumatoid Arthritis (RA) and healthy controls. Two high-density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis in order to determine patterns of signalling cascades or tissue origin. 44% of patients with Ankylosing Spondylitis demonstrated a broad autoantibody response, as compared to 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The AS patients autoantibody responses were targeted towards connective, skeletal and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic Acid Programmable Protein Arrays constitute a powerful tool to study autoimmune diseases.

After successful completion of the Human Genome Project (HGP), HUPO has recently officially launched a global Human Proteome Project (HPP) which is designed to map the entire human protein set. Given the presence of about 30% undisclosed proteins out of 20,300 protein gene products, a systematic global effort is necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP groups employ the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge base. The HPP participants will take advantage of the output and cross-analyses from the ongoing HUPO initiatives and a chromosome-based protein mapping strategy, termed C-HPP with many national teams currently engaged. In addition, numerous biologically-driven projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents and tools for protein studies and analyses, and a stronger basis for personalized medicine. HUPO urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

This article provides an introduction to Fourier transform-based mass spectrometry (FTMS). The key performance characteristics of FTMS, mass accuracy and resolution, are presented in the view of how they impact the interpretation of measurements in proteomic applications. The theory and principles of operation of two types of mass analyzer, Fourier transform ion cyclotron resonance and Orbitrap, are described. Major benefits as well as limitations of FTMS technology are discussed in the context of practical sample analysis, and illustrated with examples included as figures in this text and in the accompanying slide set. Comparisons highlighting the performance differences between the two mass analyzers are made where deemed useful in assisting the user with choosing the most appropriate technology for his/her application. Recent developments of these high-performing mass spectrometers are mentioned to provide a future outlook.

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring (PRM)-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11-18) or acquired resistance (11-18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11-18 cell model was associated not only with previously reported upregulation of MET, but also with upregulation of FLK2 and downregulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with PRM data. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706.

This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range.  Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

In bottom-up mass spectrometry-based proteomics, relative protein quantification is often achieved with data-dependent acquisition (DDA), data-independent acquisition (DIA), or selected reaction monitoring (SRM). These workflows quantify proteins by summarizing the abundances of all the spectral features of the protein (e.g., precursor ions, transitions or fragments) in a single value per protein per run. When abundances of some features are inconsistent with the overall protein profile (for technological reasons such as interferences, or for biological reasons such as post-translational modifications), the protein-level summaries and the downstream conclusions are undermined. We propose a statistical approach that automatically detects spectral features with such inconsistent patterns. The detected features can be separately investigated, and if necessary removed from the dataset. We evaluated the proposed approach on a series of benchmark controlled mixtures and biological investigations with DDA, DIA and SRM data acquisitions. The results demonstrated that it can facilitate and complement manual curation of the data. Moreover, it can improve the estimation accuracy, sensitivity and specificity of detecting differentially abundant proteins, and reproducibility of conclusions across different data processing tools. The approach is implemented as an option in the open-source R-based software MSstats.

Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity due to the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

Genetic and genomic research has greatly advanced our understanding of heart disease. Yet, comprehensive, in-depth, quantitative maps of protein expression in hearts of living humans are still lacking. Using samples obtained during valve replacement surgery in patients with mitral valve prolapse (MVP), we set out to define inter-chamber differences, the intersect of proteomic data with genetic or genomic datasets, and the impact of left atrial dilation on the proteome of patients with no history of atrial fibrillation (AF).  We collected biopsies from right atria (RA), left atria (LA) and left ventricle (LV) of seven male patients with mitral valve regurgitation with dilated LA but no history of AF. Biopsy samples were analyzed by high-resolution mass spectrometry (MS), where peptides were pre-fractionated by reverse phase high-pressure liquid chromatography prior to MS measurement on a Q-Exactive-HF Orbitrap instrument. We identified 7,314 proteins based on 130,728 peptides. Results were confirmed in an independent set of biopsies collected from three additional individuals. Comparative analysis against data from post-mortem samples showed enhanced quantitative power and confidence level in samples collected from living hearts. Our analysis, combined with data from genome wide association studies suggested candidate gene associations to MVP, identified higher abundance in ventricle for proteins associated with cardiomyopathies and revealed the dilated LA proteome, demonstrating differential representation of molecules previously associated with AF, in non-AF hearts. This is the largest dataset of cardiac protein expression from human samples collected in vivo. It provides a comprehensive resource that allows insight into molecular fingerprints of MVP and facilitates novel inferences between genomic data and disease mechanisms. We propose that over-representation of proteins in ventricle is consequent not to redundancy but to functional need, and conclude that changes in abundance of proteins known to associate with AF are not sufficient for arrhythmogenesis.

Accumulation and propagation of hyperphosphorylated tau (p-tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-tau pathology after injection into mouse brain.  To gain an understanding of the mTau exosome cargo involved in tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in (1) proteins uniquely present only in mTau, and not control exosomes, (2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and (3) shared proteins which were significantly up-regulated or down-regulated in mTau compared to control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-tau.  Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes.  Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or down-regulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-tau neuropathology in mouse brain. 

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Since these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.