The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

A Hybrid Orbitrap-Nanoelectromechanical Systems Approach for the Analysis of Individual, Intact Proteins in Real Time.

Nanoelectromechanical systems (NEMS)-based mass spectrometry (MS) is an emerging technique that enables determination of the mass of individual adsorbed particles by driving nanomechanical devices at resonance and monitoring the real-time changes in their resonance frequencies induced by each single molecule adsorption event. We incorporate NEMS into an Orbitrap mass spectrometer and report our progress towards leveraging the single-molecule capabilities of the NEMS to enhance the dynamic range of conventional MS instrumentation and to offer new capabilities for performing deep proteomic analysis of clinically relevant samples. We use the hybrid instrument to deliver E. coli GroEL molecules (801 kDa) to the NEMS devices in their native, intact state. Custom ion optics are used to focus the beam down to 40 µm diameter with a maximum flux of 25 molecules/second. The mass spectrum obtained with NEMS-MS shows good agreement with the known mass of GroEL.

Requirements and attributes of nano-resonator mass spectrometry for the analysis of intact viral particles.

When studying viruses, the most prevalent aspects that come to mind are their structural and functional features, but this leaves in the shadows a quite universal characteristic: their mass. Even if approximations can be derived from size and density measurements, the multi MDa to GDa mass range, featuring a majority of viruses, has so far remained largely unexplored. Recently, nano-electromechanical resonator-based mass spectrometry (NEMS-MS) has demonstrated the ability to measure the mass of intact DNA filled viral capsids in excess of 100 MDa. However, multiple factors have to be taken in consideration when performing NEMS-MS measurements. In this article, phenomena influencing NEMS-MS mass estimates are listed and discussed, including some particle's extraneous physical properties (size, aspect ratio, stiffness), and the influence of frequency noise and device fabrication defects. These factors being accounted for, we could begin to notice subtler effects linked with (e.g.) particle desolvation as a function of operating parameters. Graphical abstract.

Large-Scale SRM Screen of Urothelial Bladder Cancer Candidate Biomarkers in Urine.

Urothelial bladder cancer is a condition associated with high recurrence and substantial morbidity and mortality. Noninvasive urinary tests that would detect bladder cancer and tumor recurrence are required to significantly improve patient care. Over the past decade, numerous bladder cancer candidate biomarkers have been identified in the context of extensive proteomics or transcriptomics studies. To translate these findings in clinically useful biomarkers, the systematic evaluation of these candidates remains the bottleneck. Such evaluation involves large-scale quantitative LC-SRM (liquid chromatography-selected reaction monitoring) measurements, targeting hundreds of signature peptides by monitoring thousands of transitions in a single analysis. The design of highly multiplexed SRM analyses is driven by several factors: throughput, robustness, selectivity and sensitivity. Because of the complexity of the samples to be analyzed, some measurements (transitions) can be interfered by coeluting isobaric species resulting in biased or inconsistent estimated peptide/protein levels. Thus the assessment of the quality of SRM data is critical to allow flagging these inconsistent data. We describe an efficient and robust method to process large SRM data sets, including the processing of the raw data, the detection of low-quality measurements, the normalization of the signals for each protein, and the estimation of protein levels. Using this methodology, a variety of proteins previously associated with bladder cancer have been assessed through the analysis of urine samples from a large cohort of cancer patients and corresponding controls in an effort to establish a priority list of most promising candidates to guide subsequent clinical validation studies.

Correction: Predictive chromatography of peptides and proteins as a complementary tool for proteomics.

Correction for 'Predictive chromatography of peptides and proteins as a complementary tool for proteomics' by Irina A. Tarasova et al., Analyst, 2016, 141, 4816-4832.

hEIDI: An Intuitive Application Tool To Organize and Treat Large-Scale Proteomics Data.

Advances in high-throughput proteomics have led to a rapid increase in the number, size, and complexity of the associated data sets. Managing and extracting reliable information from such large series of data sets require the use of dedicated software organized in a consistent pipeline to reduce, validate, exploit, and ultimately export data. The compilation of multiple mass-spectrometry-based identification and quantification results obtained in the context of a large-scale project represents a real challenge for developers of bioinformatics solutions. In response to this challenge, we developed a dedicated software suite called hEIDI to manage and combine both identifications and semiquantitative data related to multiple LC-MS/MS analyses. This paper describes how, through a user-friendly interface, hEIDI can be used to compile analyses and retrieve lists of nonredundant protein groups. Moreover, hEIDI allows direct comparison of series of analyses, on the basis of protein groups, while ensuring consistent protein inference and also computing spectral counts. hEIDI ensures that validated results are compliant with MIAPE guidelines as all information related to samples and results is stored in appropriate databases. Thanks to the database structure, validated results generated within hEIDI can be easily exported in the PRIDE XML format for subsequent publication. hEIDI can be downloaded from http://biodev.extra.cea.fr/docs/heidi .

Efficient Exploitation of Separation Space in Two-Dimensional Liquid Chromatography System for Comprehensive and Efficient Proteomic Analyses.

Proteomics aims to achieve complete profiling of the protein content and protein modifications in cells, tissues, and biofluids and to quantitatively determine changes in their abundances. This information serves to elucidate cellular processes and signaling pathways and to identify candidate protein biomarkers and/or therapeutic targets. Analyses must therefore be both comprehensive and efficient. Here, we present a novel online two-dimensional reverse-phase/reverse-phase liquid chromatography separation platform, which is based on a newly developed online noncontiguous fractionating and concatenating device (NCFC fractionator). In bottom-up proteomics analyses of a complex proteome, this system provided significantly improved exploitation of the separation space of the two RPs, considerably increasing the numbers of peptides identified compared to a contiguous 2D-RP/RPLC method. The fully automated online 2D-NCFC-RP/RPLC system bypassed a number of labor-intensive manual processes required with the previously described offline 2D-NCFC RP/RPLC method, and thus, it offers minimal sample loss in a context of highly reproducible 2D-RP/RPLC experiments.

Predictive chromatography of peptides and proteins as a complementary tool for proteomics.

In the last couple of decades, considerable effort has been focused on developing methods for quantitative and qualitative proteome characterization. The method of choice in this characterization is mass spectrometry used in combination with sample separation. One of the most widely used separation techniques at the front end of a mass spectrometer is high performance liquid chromatography (HPLC). A unique feature of HPLC is its specificity to the amino acid sequence of separated peptides and proteins. This specificity may provide additional information about the peptides or proteins under study which is complementary to the mass spectrometry data. The value of this information for proteomics has been recognized in the past few decades, which has stimulated significant effort in the development and implementation of computational and theoretical models for the prediction of peptide retention time for a given sequence. Here we review the advances in this area and the utility of predicted retention times for proteomic applications.

Multiplexed and data-independent tandem mass spectrometry for global proteome profiling.

One of the most important early developments in the field of proteomics was the advent of automated data acquisition routines that allowed high-throughput unattended data acquisition during HPLC introduction of peptide mixtures to a tandem mass spectrometer. Prior to this, data acquisition was orders of magnitude less efficient being based entirely on lists of predetermined ions generated in a prior HPLC-MS experiment. This process, known generically as data-dependent analysis, empowered the development of shotgun proteomics where hundreds to thousands of peptide sequences are matched per experiment. In their most popular implementation, the most abundant ionized species from every precursor ion scan at each moment in chromatographic time are successively selected for isolation, activation and tandem mass analysis. While extremely powerful, this strategy has one primary limitation in that detectable dynamic range is restricted (in a top-down manner) to the peptides that ionize the best. To circumvent the serial nature of the data-dependent process and increase detectable dynamic range, the concepts of multiplexed and data-independent acquisition (DIA) have emerged. Multiplexed-data acquisition is based on more efficient co-selection and co-dissociation of multiple precursor ions in parallel, the data from which is subsequently de-convoluted to provide polypeptide sequences for each individual precursor ion. DIA has similar goals, but there is no real-time ion selection based on prior precursor ion scans. Instead, predefined m/z ranges are interrogated either by fragmenting all ions entering the mass spectrometer at every single point in chromatographic time; or by dividing the m/z range into smaller m/z ranges for isolation and fragmentation. These approaches aim to fully utilize the capabilities of mass spectrometers to maximize tandem MS acquisition time and to address the need to expand the detectable dynamic range, lower the limit of detection, and improve the overall confidence of peptide identifications and relative protein quantification measurements. This review covers all aspects of multiplexed- and data-independent tandem mass spectrometry in proteomics, from experimental implementations to advances in software for data interpretation.

Compact and modular system architecture for a nano-resonator-mass spectrometer.

Mass measurements in the mega-to giga-Dalton range are essential for the characterization of natural and synthetic nanoparticles, but very challenging to perform using conventional mass spectrometers. Nano-electro-mechanical system (NEMS) based MS has demonstrated unique capabilities for the analysis of ultra-high mass analytes. Yet, system designs to date included constraints transferred from conventional MS instruments, such as ion guides and high vacuum requirements. Encouraged by other reports, we investigated the influence of pressure on the performances of the NEMS sensor and the aerodynamic focusing lens that equipped our first-generation instrument. We thus realized that the NEMS spectrometer could operate at significantly higher pressures than anticipated without compromising particle focusing nor mass measurement quality. Based on these observations, we designed and constructed a new NEMS-MS prototype considerably more compact than our original system, and which features an improved aerodynamic lens alignment concept, yielding superior particle focusing. We evaluated this new prototype by performing nanoparticle deposition to characterize aerodynamic focusing, and mass measurements of calibrated gold nanoparticles samples. The particle capture efficiency showed nearly two orders of magnitude improvement compared to our previous prototype, while operating at two orders of magnitude greater pressure, and without compromising mass resolution.

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry.

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.

Surface acoustic wave nebulization facilitating lipid mass spectrometric analysis.

Surface acoustic wave nebulization (SAWN) is a novel method to transfer nonvolatile analytes directly from the aqueous phase to the gas phase for mass spectrometric analysis. The lower ion energetics of SAWN and its planar nature make it appealing for analytically challenging lipid samples. This challenge is a result of their amphipathic nature, labile nature, and tendency to form aggregates, which readily precipitate clogging capillaries used for electrospray ionization (ESI). Here, we report the use of SAWN to characterize the complex glycolipid, lipid A, which serves as the membrane anchor component of lipopolysaccharide (LPS) and has a pronounced tendency to clog nano-ESI capillaries. We also show that unlike ESI SAWN is capable of ionizing labile phospholipids without fragmentation. Lastly, we compare the ease of use of SAWN to the more conventional infusion-based ESI methods and demonstrate the ability to generate higher order tandem mass spectral data of lipid A for automated structure assignment using our previously reported hierarchical tandem mass spectrometry (HiTMS) algorithm. The ease of generating SAWN-MS(n) data combined with HiTMS interpretation offers the potential for high throughput lipid A structure analysis.

Identification of cellular targets in human intrahepatic cholangiocarcinoma using laser microdissection and accurate mass and time tag proteomics.

Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Toward a standardized urine proteome analysis methodology.

Urine is an easily accessible bodily fluid particularly suited for the routine clinical analysis of disease biomarkers. Actually, the urinary proteome is more diverse than anticipated a decade ago. Hence, significant analytical and practical issues of urine proteomics such as sample collection and preparation have emerged, in particular for large-scale studies. We have undertaken a systematic study to define standardized and integrated analytical protocols for a biomarker development pipeline, employing two LC-MS analytical platforms, namely accurate mass and time tags and selected reaction monitoring, for the discovery and verification phase, respectively. Urine samples collected from hospital patients were processed using four different protocols, which were evaluated and compared on both analytical platforms. Addition of internal standards at various stages of sample processing allowed the estimation of protein extraction yields and the absolute quantification of selected urinary proteins. Reproducibility of the entire process and dynamic range of quantification were also evaluated. Organic solvent precipitation followed by in-solution digestion provided the best performances and was thus selected as the standard method common to the discovery and verification phases. Finally, we applied this protocol for platforms' cross-validation and obtained excellent consistency between urinary protein concentration estimates by both analytical methods performed in parallel in two laboratories.

A toolbox for validation of mass spectrometry peptides identification and generation of database: IRMa.

SUMMARY: The IRMa toolbox provides an interactive application to assist in the validation of Mascot search results. It allows automatic filtering of Mascot identification results as well as manual confirmation or rejection of individual PSM (a match between a fragmentation mass spectrum and a peptide). Dynamic grouping and coherence of information are maintained by the software in real time. Validated results can be exported under various forms, including an identification database (MSIdb). This allows biologists to compile search results from a whole study in a unique repository in order to provide a summarized view of their project. IRMa also features a fully automated version that can be used in a high-throughput pipeline. Given filter parameters, it can delete hits with no significant PSM, regroup hits identified by the same peptide(s) and export the result to the specified format without user intervention. AVAILABILITY: http://biodev.extra.cea.fr/docs/irma (java 1.5 or higher needed).

Optomechanical mass spectrometry.

Nanomechanical mass spectrometry has proven to be well suited for the analysis of high mass species such as viruses. Still, the use of one-dimensional devices such as vibrating beams forces a trade-off between analysis time and mass resolution. Complex readout schemes are also required to simultaneously monitor multiple resonance modes, which degrades resolution. These issues restrict nanomechanical MS to specific species. We demonstrate here single-particle mass spectrometry with nano-optomechanical resonators fabricated with a Very Large Scale Integration process. The unique motion sensitivity of optomechanics allows designs that are impervious to particle position, stiffness or shape, opening the way to the analysis of large aspect ratio biological objects of great significance such as viruses with a tail or fibrils. Compared to top-down beam resonators with electrical read-out and state-of-the-art mass resolution, we show a three-fold improvement in capture area with no resolution degradation, despite the use of a single resonance mode.

Protein Biomarker Discovery in Non-depleted Serum by Spectral Library-Based Data-Independent Acquisition Mass Spectrometry.

In discovery proteomics experiments, tandem mass spectrometry and data-dependent acquisition (DDA) are classically used to identify and quantify peptides and proteins through database searching. This strategy suffers from known limitations such as under-sampling and lack of reproducibility of precursor ion selection in complex proteomics samples, leading to somewhat inconsistent analytical results across large datasets. Data-independent acquisition (DIA) based on fragmentation of all the precursors detected in predetermined isolation windows can potentially overcome this limitation. DIA promises reproducible peptide and protein quantification with deeper proteome coverage and fewer missing values than DDA strategies. This approach is particularly attractive in the field of clinical biomarker discovery, where large numbers of samples must be analyzed. Here, we describe a DIA workflow for non-depleted serum analysis including a straightforward approach through which to construct a dedicated spectral library, and indications on how to optimize chromatographic and mass spectrometry analytical methods to produce high-quality DIA data and results.

Prospects for monolithic nano-LC columns in shotgun proteomics.

We report a premier side-by-side comparison of two leading types of monolithic nano-LC column (silica-C(18), polystyrene) in shotgun proteomics experiments. Besides comparing the columns in terms of the number of peptides from a real-life sample (Arabidopsis thaliana chloroplast) that they identified, we compared the monoliths in terms of peak capacity and retention behavior for standard peptides. For proteomics applications where the mobile phase composition is constrained by electrospray ionization considerations (i.e., there is a restricted choice of ion-pairing modifiers), the polystyrene nano-LC column exhibited reduced identification power. The silica monolith column was superior in all measured values and compared very favorably with traditional packed columns. Finally, we investigated the performances of the monoliths at high flow rates in an attempt to demonstrate their advantages for high-throughput identification.

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators.

Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.