Search

Antibody mediated strategies for protein biomarker detection are common, but may limit discovery. We hypothesized that the use of antibody-free proteomics is feasible for detecting protein biomarkers in plasma of patients sustaining major trauma. A subset of subjects with major trauma from a prospective observational trial were analyzed. Patients were assigned to one of four groups based on their presenting Abbreviated Injury Severity Score (AIS). Sensitive, antibody-free selective reaction monitoring (SRM) mass spectrometry (MS), with spiked-in isotopically labeled synthetic peptides, was used for targeted protein quantification of a panel of 10 prospective targets. An overall tiered sensitivity analytical approach was used for peptide detection and quantification based upon plasma immunoaffinity depletion and PRISM fractionation. Forty-four patients were included in the analysis, of which 82% were men with a mean age of 50 (±19) years. Half had isolated head injury (n = 22), with the remaining patients experiencing multiple injuries or polytrauma (n = 14), isolated body injury (n = 2), or minor injury (n = 6). Peptides from 3 proteins (vascular adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and matrix metalloproteinase 9 [MMP9]) were detected and quantified in non-depleted processed plasma. Peptides from 2 proteins (angiopoietin 2 [Ang2] and plasminogen activator inhibitor-1 [PAI1]) were detected and quantification in depleted plasma, whereas the remaining 5 of the 10 prospective targets were undetected. VCAM1 (p = 0.02) and MMP9 (p = 0.03) were significantly upregulated in in the major trauma groups (1–3) versus mild injury group (4), whereas the others were not. There were no differences in protein expression between patients with traumatic brain injury (TBI; groups 1 and 2) versus those without TBI (groups 3 and 4). We detected non-specific upregulation of proteins reflecting blood–brain barrier breakdown in severely injured patients, indicating label-free MS techniques are feasible and may be informative.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics., Tsai, Zhang, Scholten et al. develop a surfactant- assisted one-pot sample preparation coupled with mass spectrometry method (SOP-MS) for label-free global single-cell proteomics. This method allows researchers to measure hundreds of proteins from single human cells, suggesting its utility for quantitative single-cell proteomics.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt., Habowski et al. develop a sorting protocol for purification and comparative analysis of mouse colon stem cells and their progeny. By proteomic and transcriptomic analysis, they determine that lineage commitment is accompanied by a greater change in mRNA splicing and polyadenylation than in gene expression and they characterise signalling pathways involved in differentiation.

Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS ( s urfactant-assisted o ne- p ot sample processing at the s tandard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low μL volume, we have systematically evaluated its processing volume at 10-200 μL using 100 human cells. The processing volume of 50 μL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200-2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, whereas they have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative in terms of being antibody-free, high multiplex, high specificity, and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) – selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses and high-specificity LC-SRM to effectively address the increased dynamic concentration range resulted from the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement feature and no need of specific devices make this method readily accessible to most proteomics laboratories. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

Protein phosphorylation is a critical post-translational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement, and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.

Protein analysis of small numbers of human cells and single cells is primarily achieved by targeted proteomics with antibody-based immunoassays, whereas they have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative in terms of being antibody-free, high multiplex, high specificity, and quantitation accuracy. Recent advances in MS instrumentation makes MS-based targeted single-cell proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical issue for effective processing of single cells with minimal sample loss for MS analysis. To address this issue we have recently developed a simple protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) – selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells including single cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses and high-specificity LC-SRM to effectively address the increased dynamic concentration range resulted from the addition of exogeneous carrier protein. Herein we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. cLC-SRM was demonstrated to enable multiplexed accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10–100 cells) and highly abundant proteins in single cells. Most importantly, this method can be easily implemented in any MS and proteomics laboratories at no additional cost for instrument or reagents. Further improvements in detection sensitivity and sample throughput are needed towards multiplexed targeted single-cell proteomics analysis. We anticipate that eventually it can be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.