Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics.

Protein identification and quantification is an important tool for biomarker discovery. With the increased sensitivity and speed of modern mass spectrometers, sample preparation remains a bottleneck for studying large cohorts. To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.

One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes.

Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types.

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation.

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

Development of a Standardized Microflow LC Gradient to Enable Sensitive and Long-Term Detection of Synthetic Anabolic-Androgenic Steroids for High-Throughput Doping Controls.

Synthetic androgenic anabolic steroids (AAS) are banned compounds and considered as major threats by both racing and sports international authorities. Hence, doping control laboratories are continually looking into analytical improvements to increase their detection capabilities, notably by means of emerging technologies. To enhance analytical performances for the detection of synthetic AAS such as stanozolol, specific chromatographic procedures have been developed using recent quaternary liquid chromatography technology originally designed for high-throughput standardized proteomics connected to mass spectrometry. Applying the newly designed elution procedures described in this paper to the analyses of stanozolol and its metabolites in complex matrixes revealed improved sensitivity compared to previously described high-throughput methods. Indeed, we report the consistent and reliable detection of 16ß-hydroxy-stanozolol down to 10 pg/mL in equine urine and being detectable up-to 3 months after a microdosing administration. Furthermore, a five months long elimination of stanozolol and its metabolites could be monitored on horse mane sections after a single dose administration. Our work highlights novel solutions to detect AAS with improved sensitivity. The application of such developments constitutes new landmarks for doping control laboratories and could be extended to other targeted compounds in residue analysis, toxicology, and metabolomics. Based on this work, the developed chromatographic method is now freely available within the Evosep Plus program.

diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

Evosep One Enables Robust Deep Proteome Coverage Using Tandem Mass Tags while Significantly Reducing Instrument Time.

The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12 000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.

Online Parallel Accumulation-Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer.

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics.

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are performed in parallel, reducing overhead time to a few minutes and allowing analysis of more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering more than 130,000 sequence unique peptides and close to 10,000 proteins were rapidly acquired. Using this data as a library, we demonstrate quantitation of 5200 proteins in only 21 min. Thus, the new system - termed Evosep One - analyzes samples in an extremely robust and high throughput manner, without sacrificing in depth proteomics coverage.

Detection and differentiation of 22 kDa and 20 kDa Growth Hormone proteoforms in human plasma by LC-MS/MS.

Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20 kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20 kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation.

Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7µL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples for reduced signal suppression and increased sensitivity in LC-MS/MS. We benchmarked this method for the analysis of 40 individual blood samples including 20 patients diagnosed with breast cancer. BIOLOGICAL SIGNIFICANCE: The interest for MS-based biomarker analysis in body fluids is steadily increasing as proteomics methodology translates into clinical laboratories. We describe a method for detection of 5 distinct proteins of low mass and medium to low abundance, which are of interest in anti-doping and clinical analysis. The analytical setup is simple and robust and is suitable for high-throughput instrument configurations.

Rapid analyses of proteomes and interactomes using an integrated solid-phase extraction-liquid chromatography-MS/MS system.

Here, we explore applications of a LC system using disposable solid-phase extraction (SPE) cartridges and very short LC-MS/MS gradients that allows for rapid analyses in less than 10 min analysis time. The setup consists of an autosampler harboring two sets of 96 STAGE tips that function as precolumns and a short RP analytical column running 6.5 min gradients. This system combines efficiently with several proteomics workflows such as offline prefractionation methods, including 1D gel electrophoresis and strong-cation exchange chromatography. It also enables the analysis of interactomes obtained by affinity purification with an analysis time of approximately 1 h. In a typical shotgun proteomics experiment involving 36 SCX fractions of an AspN digested cell lysate, we detected over 3600 protein groups with an analysis time of less than 5.5 h. This innovative fast LC system reduces proteome analysis time while maintaining sufficient proteomic detail. This has particular relevance for the use of proteomics within a clinical environment, where large sample numbers and fast turnover times are essential.

Integrated solid-phase extraction-capillary liquid chromatography (speLC) interfaced to ESI-MS/MS for fast characterization and quantification of protein and proteomes.

The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enables the utilization of fast liquid chromatographic (LC) separation techniques. We present a robust solid-phase extraction/capillary LC system (speLC) for 5-10 min separation of semicomplex peptide mixtures prior to ESI-MS/MS for peptide sequencing. This speLC-MS/MS system eliminates sample-to-sample carry-over by using disposable micropipette solid-phase extraction tips (StageTips) for peptide sample loading, concentration, and desalting. Automated analysis of 192 replicates of E. coli peptide mixtures in 30 h demonstrated the throughput, stability, and reproducibility of the system. The speLC-MS/MS system detected low-femtomole amounts of peptides and allowed sequencing of 1 µg of HeLa cells protein extracts at a rate of ∼ 90 peptides/min, identifying more than 1500 peptides (>500 proteins) in a 10 min speLC-MS/MS experiment. Analysis by selected reaction monitoring by speLC-SRM-MS/MS of distinct peptides derived from the blood proteins IGF1, IGF2, IBP2, and IBP3 demonstrated protein quantification with CV values below 10% across 96 replicates. The speLC-MS/MS system is ideally suited for fast screening and characterization of large numbers of peptide-containing samples in biological, biomedical, and clinical laboratories.

Characterization and usage of the EASY-spray technology as part of an online 2D SCX-RP ultra-high pressure system.

Ultra-high pressure liquid chromatography (UHPLC) systems combined with state-of-the-art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC-MS experiment within a few hours. The recently released EASY-spray technology allows one to implement nano-UHPLC with straightforwardness. In this work we initially characterized the EASY-spray containing a 50 cm column containing <2 µm particles and found that the system allowed 3000 proteins to be identified in 90 minutes. We then asked the question whether a fast and sensitive online 2D SCX-RP UHPLC-MS/MS workflow could compete with 1D long gradient analyses, using total analysis time versus proteome coverage and sample usage as benchmark parameters. The 2D LC-MS strategy consisted of the EASY-spray system that had been augmented by the addition of an SCX column. The conversion was made facile since no additional valves were required and by the use of components containing viper fittings. We benchmarked the system using a human cell lysate digest (<10 µg). The 2D SCX-RP UHPLC-MS/MS workflow allowed the identification of almost 37,000 unique peptides and 6000 proteins in a total analysis time of ~7 hours. On the same system a 1D RP UHPLC-MS/MS workflow plateaued at only 20,000 peptides and 4400 unique proteins and required approx. 8 hours of analysis time. Furthermore, the 2D workflow could continue to increase the proteome coverage with longer analysis times, in fact with a 21 hour analysis we identified 56,600 unique peptides and >7500 proteins. We report, here, that with this fast online SCX-RP UHPLC-MS/MS workflow, the proteome coverage can be substantially extended without significantly compromising the analysis time and sample usage.

Spatially resolved protein hydrogen exchange measured by matrix-assisted laser desorption ionization in-source decay.

Mass spectrometry has become a powerful tool for measuring protein hydrogen exchange and thereby reveal the structural dynamics of proteins in solution. Here we describe the successful application of a matrix-assisted laser desorption ionization (MALDI) mass spectrometry approach based on in-source decay (ISD) to measure spatially resolved amide backbone hydrogen exchange. By irradiating deuterated protein molecules in a crystalline matrix with a high laser fluence, they undergo prompt fragmentation. Spatially resolved deuteration levels are readily obtained by mass analysis of consecutive fragment ions. MALDI ISD analysis of deuterated cytochrome c yielded an extensive series of c-fragment ions which originate from cleavage of nearly all N-C(α) bonds (Cys17 to Glu104) allowing for a detailed analysis of the deuterium content of the backbone amides. While hydrogen scrambling can be major concern when using mass spectrometric fragmentation to obtain detailed information on protein hydrogen exchange, we show that the level of hydrogen scrambling in our MALDI ISD measurements is negligible and that the known dynamic behavior of cytochrome c in solution is accurately reflected in the deuterium contents of the fragment ions. The developed method combines several attractive features from a practical point of view as it is simple to perform and it readily provides a detailed mapping of the dynamic structure of a protein in solution.

Identification of nitrotyrosine containing peptides using combined fractional diagonal chromatography (COFRADIC) and off-line nano-LC-MALDI.

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.

A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation.

Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved in the assembly of clathrin coated vesicles in synaptic vesicle endocytosis. Unlike other types of O-glycosylation, O-GlcNAc is nucleocytoplasmic and reversible. It was thought to be a terminal modification, that is, the O-GlcNAc was not found to be additionally modified in any way. We now show that AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification.

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles.

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.

Biomarker candidate discovery in Atlantic cod (Gadus morhua) continuously exposed to North Sea produced water from egg to fry.

In this study Atlantic cod (Gadus morhua) were exposed to different levels of North Sea produced water (PW) and 17beta-oestradiol (E(2)), a natural oestrogen, from egg to fry stage (90 days). By comparing changes in protein expression following E(2) exposure to changes induced by PW treatment, we were able to compare the induced changes by PW to the mode of action of oestrogens. Changes in the proteome in response to exposure in whole cod fry (approximately 80 days post-hatching, dph) were detected by two-dimensional gel electrophoresis and image analysis and identified by MALDI-ToF-ToF mass spectrometry, using a newly developed cod EST database and the NCBI database. Many of the protein changes occurred at low levels (0.01% and 0.1% PW) of exposure, indicating putative biological responses at lower levels than previously detected. Using discriminant analysis, we identified a set of protein changes that may be useful as biomarker candidates of produced water (PW) and oestradiol exposure in Atlantic cod fry. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring exposure and effects of discharges from the petroleum industry offshore, aiding future environmental risk analysis and risk management.

Proteomics of the photoneuroendocrine circadian system of the brain.

The photoneuroendocrine circadian system of the brain consists of (a) specialized photoreceptors in the retina, (b) a circadian generator located in the forebrain that contains "clock genes," (c) specialized nuclei in the forebrain involved in neuroendocrine secretion, and (d) the pineal gland. The circadian generator is a nucleus, called the suprachiasmatic nucleus (SCN). The neurons of this nucleus contain "clock genes," the transcription of which exhibits a circadian rhythm. Most circadian rhythms are generated by the neurons of this nucleus and, via neuronal and humoral connections, the SCN controls circadian activity of the brain and peripheral tissues. The endogenous oscillator of the SCN is each day entrained to the length of the daily photoperiod by light that reach the retina, and specialized photoreceptors transmit impulses to the SCN via the optic nerves. Mass screening for day/night variations in gene expression in the circadian system as well as in the whole brain and peripheral tissues have, during the last decade, been performed. However, studies of circadian changes in the proteome have been less investigated. In this survey, the anatomy and function of the circadian-generating system in mammals is described, and recent proteomic studies that investigate day/night changes in the retina, SCN, and pineal gland are reviewed. Further circadian changes controlled by the SCN in gene and protein expression in the liver are discussed.