Easy and Accessible Workflow for Label-Free Single-Cell Proteomics.

Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often requires custom instrumentation, consumables, and reagents for sample processing and separations, which has limited the accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these platforms and the technical expertise required for their operation place them out of reach of many interested laboratories. Here, we assessed the new HP D100 Single Cell Dispenser for label-free SCP. The low-cost instrument proved highly accurate and reproducible for dispensing reagents in the range from 200 nL to 2 µL. We used the HP D100 to isolate and prepare single cells for SCP within 384-well PCR plates. When the well plates were immediately centrifuged following cell dispensing and again after reagent dispensing, we found that ∼97% of wells that were identified in the instrument software as containing a single cell indeed provided the proteome coverage expected of a single cell. This commercial dispenser combined with one-step sample processing provides a very rapid and easy-to-use workflow for SCP with no reduction in proteome coverage relative to a nanowell-based workflow, and the commercial well plates also facilitate autosampling with unmodified instrumentation. Single-cell samples were analyzed using home-packed 30 µm i.d. nanoLC columns as well as commercially available 50 µm i.d. columns. The commercial columns resulted in ∼35% fewer identified proteins. However, combined with the well plate-based preparation platform, the presented workflow provides a fully commercial and relatively low-cost alternative for SCP sample preparation and separation, which should greatly broaden the accessibility of SCP to other laboratories.

Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics.

Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive and lead to long sample-to-answer times. Here we report a sample preparation method that achieves cell lysis, protein denaturation, and digestion in 1 h using commercially available high-temperature-stabilized proteases with a single reagent dispensing step. Four different one-step reagent compositions were evaluated, and the mixture providing the highest proteome coverage was compared to the previously employed multistep workflow. The one-step preparation increases proteome coverage relative to the previous multistep workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene chips and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled the identification of an average of nearly 2400 proteins per cell using a standard data-dependent workflow with Orbitrap mass spectrometers. These advances greatly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.

Combining offline high performance liquid chromatography fractionation of peptides and intact proteins to enhance proteome coverage in bottom-up proteomics.

Offline peptide separation (PS) using high-performance liquid chromatography (HPLC) is currently used to enhance liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection of proteins. In search of more effective methods for enhancing MS proteome coverage, we developed a robust method for intact protein separation (IPS), an alternative first-dimension separation technique, and explored additional benefits that it offers. Comparing IPS to the traditional PS method, we found that both enhance detection of unique protein IDs to a similar magnitude, though in diverse ways. IPS was especially effective in serum, which has a small number of extremely high abundance proteins. PS was more effective in tissues with fewer dominating high-abundance proteins and was more effective in enhancing detection of post-translational modifications (PTMs). Combining the IPS and PS methods together (IPS+PS) was especially beneficial, enhancing proteome detection more than either method could independently. The comparison of IPS+PS versus six PS fractionation pools increased total number of proteins IDs by nearly double, while also significantly increasing number of unique peptides detected per protein, percent peptide sequence coverage of each protein, and detection of PTMs. This IPS+PS combined method requires fewer LC-MS/MS runs than current PS methods would need to obtain similar improvements in proteome detection, and it is robust, time- and cost-effective, and generally applicable to various tissue and sample types.

Modification of the structural stability of human serum albumin in rheumatoid arthritis.

Differential scanning calorimetry (DSC) can indicate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes result from either concentration changes or altered thermal stabilities for 7-10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA and the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes observed in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of some fraction of the human serum albumin (HSA) proteins in the sample. By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.

CHalf: Folding Stability Made Simple.

The structure of a protein defines its function and integrity and correlates with the protein folding stability (PFS). Quantifying PFS allows researchers to assess differential stability of proteins in different disease or ligand binding states, providing insight into protein efficacy and potentially serving as a metric of protein quality. There are a number of mass spectrometry (MS)-based methods to assess PFS, such as Thermal Protein Profiling (TPP), Stability of Proteins from Rates of Oxidation (SPROX), and Iodination Protein Stability Assay (IPSA). Despite the critical value that PFS studies add to the understanding of mechanisms of disease and treatment development, proteomics research is still primarily dominated by concentration-based studies. We found that a major reason for the lack of PFS studies is the lack of a user-friendly data processing tool. Here we present the first user-friendly software, CHalf, with a graphical user interface for calculating PFS. Besides calculating site-specific PFS of a given protein from chemical denature folding stability assays, CHalf is also compatible with thermal denature folding stability assays. CHalf also includes a set of data visualization tools to help identify changes in PFS across protein sequences and in between different treatment conditions. We expect the introduction of CHalf to lower the barrier of entry for researchers to investigate PFS, promoting the usage of PFS in studies. In the long run, we expect this increase in PFS research to accelerate our understanding of the pathogenesis and pathophysiology of disease.

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase cassettes, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of structures of Cdc48 affinity purified from lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data reveal new elements of Shp1-Cdc48 binding and support a "hand-over-hand" mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by D2 in substrate translocation/unfolding.

Quantifying In Situ Structural Stabilities of Human Blood Plasma Proteins Using a Novel Iodination Protein Stability Assay.

Many of the diseases that plague society today are driven by a loss of protein quality. One method to quantify protein quality is to measure the protein folding stability (PFS). Here, we present a novel mass spectrometry (MS)-based approach for PFS measurement, iodination protein stability assay (IPSA). IPSA quantifies the PFS by tracking the surface-accessibility differences of tyrosine, histidine, methionine, and cysteine under denaturing conditions. Relative to current methods, IPSA increases protein coverage and granularity to track the PFS changes of a protein along its sequence. To our knowledge, this study is the first time the PFS of human serum proteins has been measured in the context of the blood serum (in situ). We show that IPSA can quantify the PFS differences between different transferrin iron-binding states in near in vivo conditions. We also show that the direction of the denaturation curve reflects the in vivo surface accessibility of the amino acid residue and reproducibly reports a residue-specific PFS. Along with IPSA, we introduce an analysis tool Chalf that provides a simple workflow to calculate the residue-specific PFS. The introduction of IPSA increases the potential to use protein structural stability as a structural quality metric in understanding the etiology and progression of human disease. Data is openly available at Chorusproject.org (project ID 1771).

Proposing a minimal set of metrics and methods to predict probabilities of amyloidosis disease and onset age in individuals.

Many amyloid-driven pathologies have both genetic and stochastic components where assessing risk of disease development requires a multifactorial assessment where many of the variables are poorly understood. Risk of transthyretin-mediated amyloidosis is enhanced by age and mutation of the transthyretin (TTR) gene, but amyloidosis is not directly initiated by mutated TTR proteins. Nearly all of the 150+ known mutations increase dissociation of the homotetrameric protein structure and increase the probability of an individual developing a TTR amyloid disease late in life. TTR amyloidosis is caused by dissociated monomers that are destabilized and refold into an amyloidogenic form. Therefore, monomer concentration, monomer proteolysis rate, and structural stability are key variables that may determine the rate of development of amyloidosis. Here we develop a unifying biophysical model that quantifies the relationships among these variables in plasma and suggest the probability of an individual developing a TTR amyloid disease can be estimated. This may allow quantification of risk for amyloidosis and provide the information necessary for development of methods for early diagnosis and prevention. Given the similar observation of genetic and sporadic amyloidoses for other diseases, this model and the measurements to assess risk may be applicable to more proteins than just TTR.

CORP: The use of deuterated water for the measurement of protein synthesis.

The use of deuterium oxide (D2O) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of D2O labeling is that it facilitates the measurement of synthesis rates over prolonged periods of time from single proteins through integrated tissue-based measurements. Because the ease of administration, the method is amenable for use in a variety of models and conditions. Although the method adheres to the same rules as other isotope methods, the flexibility can create conditions that are not the same as other approaches and thus requires careful execution to maintain validity and reliability. For this CORP article, we provide a history that gave rise to the method and discuss the advantages and disadvantages of the method, the critical assumptions, guidelines, and best practices based on instrumentation, models, and experimental design. The goal of this CORP article is to propagate additional use of D2O in a manner that produces reliable and valid data.

Analysis of thrombin-antithrombin complex formation using microchip electrophoresis and mass spectrometry.

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk.