We would like to acknowledge the Fall 2021 NSF I-Corps Program (Northeastern University site) award that has funded this publication.

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+cell+variability+and+drug-induced+reprogramming+as+a+mode+of+cancer+drug+resistance.">Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance.</a> Nature. 555 (7695), 274 (2018).</li><li>Pan, D., Jia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+single-cell+multi-omics+in+dissecting+cancer+cell+plasticity+and+tumor+heterogeneity.">Application of single-cell multi-omics in dissecting cancer cell plasticity and tumor heterogeneity.</a> Frontiers in Molecular Biosciences. 8, 757024 (2021).</li><li>Segerstolpe, &. #. 1. 9. 7. ;., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptome+profiling+of+human+pancreatic+islets+in+health+and+type+2+diabetes.">Single-cell transcriptome profiling of human pancreatic islets in health and type 2 diabetes.</a> Cell Metabolism. 24 (4), 593-607 (2016).</li><li>Tritschler, S., Theis, F. J., Lickert, H., B&#246;ttcher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+single-cell+analysis+provides+new+insights+into+heterogeneity+and+plasticity+of+the+pancreas.">Systematic single-cell analysis provides new insights into heterogeneity and plasticity of the pancreas.</a> Molecular Metabolism. 6 (9), 974-990 (2017).</li><li>Hanna, S. J., Tatovic, D., Thayer, T. C., Dayan, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+from+single+cell+rna+sequencing+into+the+immunology+of+type+1+diabetes-+cell+phenotypes+and+antigen+specificity.">Insights from single cell rna sequencing into the immunology of type 1 diabetes- cell phenotypes and antigen specificity.</a> Frontiers in Immunology. 12, 751701 (2021).</li><li>Baron, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+transcriptomic+map+of+the+human+and+mouse+pancreas+reveals+inter-+and+intra-cell+population+structure.">A single-cell transcriptomic map of the human and mouse pancreas reveals inter- and intra-cell population structure.</a> Cell Systems. 3 (4), 346-360 (2016).</li><li>Franks, A., Airoldi, E., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+across+human+tissues.">Post-transcriptional regulation across human tissues.</a> PLoS Computational Biology. 13 (5), 1005535 (2017).</li><li>Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpicking+the+proteome+in+single+cells.">Unpicking the proteome in single cells.</a> Science. 367 (6477), 512-513 (2020).</li><li>Specht, H., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformative+opportunities+for+single-cell+proteomics.">Transformative opportunities for single-cell proteomics.</a> Journal of Proteome Research. 17 (8), 2565-2571 (2018).</li><li>Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+from+natural+variation+across+the+proteomes+of+single+cells.">Learning from natural variation across the proteomes of single cells.</a> PLoS Biology. , (2021).</li><li>Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Driving+single+cell+proteomics+forward+with+innovation.">Driving single cell proteomics forward with innovation.</a> Journal of Proteome Research. 20 (11), 4915-4918 (2021).</li><li>Specht, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+proteomic+and+transcriptomic+analysis+of+macrophage+heterogeneity+using+SCoPE2.">Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.</a> Genome Biology. 22 (1), 50 (2021).</li><li>Petelski, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+single-cell+proteomics+using+SCoPE2.">Multiplexed single-cell proteomics using SCoPE2.</a> Nature Protocols. 16 (12), 5398-5425 (2021).</li><li>Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+single-cell+proteomics.">Scaling up single-cell proteomics.</a> Molecular and Cellular Proteomics: MCP. 21 (1), 100179 (2022).</li><li>Cong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+single-cell+proteomics+workflow+identifies&gt;+1000+protein+groups+per+mammalian+cell.">Ultrasensitive single-cell proteomics workflow identifies&gt; 1000 protein groups per mammalian cell.</a> Chemical Science. 12 (3), 1001-1006 (2021).</li><li>Lombard-Banek, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+subcellular+mass+spectrometry+enables+proteo-metabolomic+single-cell+systems+biology+in+a+chordate+embryo+developing+to+a+normally+behaving+tadpole+(X.+laevis).">In vivo subcellular mass spectrometry enables proteo-metabolomic single-cell systems biology in a chordate embryo developing to a normally behaving tadpole (X. laevis).</a> Angewandte Chemie. 60 (23), 12852-12858 (2021).</li><li>Specht, H., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+accuracy+and+depth+of+protein+quantification+in+experiments+using+isobaric+carriers.">Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers.</a> Journal of Proteome Research. 20 (1), 880-887 (2020).</li><li>Budnik, B., Levy, E., Harmange, G., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCoPE-MS:+mass+spectrometry+of+single+mammalian+cells+quantifies+proteome+heterogeneity+during+cell+differentiation.">SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation.</a> Genome Biology. 19 (1), 161 (2018).</li><li>Specht, H., Harmange, G., Perlman, D. H., Emmott, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+sample+preparation+for+high-throughput+single-cell+proteomics.">Automated sample preparation for high-throughput single-cell proteomics.</a> BioRxiv. , (2018).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+resolving+proteomes+in+single+cells.">Towards resolving proteomes in single cells.</a> Nature Methods. 18 (8), 856 (2021).</li><li>Huffman, R. G., Chen, A., Specht, H., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DO-MS:+Data-driven+optimization+of+mass+spectrometry+methods.">DO-MS: Data-driven optimization of mass spectrometry methods.</a> Journal of Proteome Research. 18 (6), 2493-2500 (2019).</li><li>Chen, A. T., Franks, A., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DART-ID+increases+single-cell+proteome+coverage.">DART-ID increases single-cell proteome coverage.</a> PLoS Computational Biology. 15 (7), 1007082 (2019).</li><li>Cheung, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+carrier+proteome+limit+for+single-cell+proteomics.">Defining the carrier proteome limit for single-cell proteomics.</a> Nature Methods. 18 (1), 76-83 (2021).</li><li>Leduc, A., Huffman, R. G., Slavov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+sample+preparation+for+single-cell+proteomics+applied+to+the+cell+cycle.">Droplet sample preparation for single-cell proteomics applied to the cell cycle.</a> bioRxiv. , (2021).</li><li>Vanderaa, C., Gatto, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+Scp+for+the+analysis+and+replication+of+single-cell+proteomics+data.">Utilizing Scp for the analysis and replication of single-cell proteomics data.</a> bioRxiv. , (2021).</li></ol>

The authors have no conflicts of interest.

One of the keys to successful preparation and analysis of single cells by SCoPE2 is the preparation of the carrier and reference channels. The suggested carrier size is 100 to 200 cells; however, the number of cells needed for a particular single-cell experiment can be determined based on principles as discussed elsewhere21. For the suggested size, at least ~11,275 cells are needed per 384-well plate, allowing for a 200-cell carrier and 5-cell reference in each set. It can be advantageous to isolate more cells if the desired cell populations are not a limiting factor, to simply have extra material in case of mistakes (as was done in this protocol, isolating 22,000 cells instead of 11,275 cells). The amount of carrier and reference needed for the number of desired plates should be prepared in a single batch to minimize any batch-to-batch variation that can cause peptide identification or quantification to differ between batches.
Before isolating single-cells into wells of a 384-well plate, it is important to consider the design of the plate layout. Within each 384-well plate, it is recommended to implement both positive and negative controls. Negative controls are wells in which no cells are added but undergo the same reagent additions and procedures as the single-cell wells. Positive controls are wells in which cell lysate diluted to 2-5 cells/&#181;L is added instead of single cells. This is particularly important to implement when single-cell isolation methods are not well-validated. Each 384-well plate should ideally have a randomized distribution of single cells and controls (e.g., not have negative or positive controls in just one row). Each plate should have an equal distribution of all cell populations of interest, rather than isolating one cell type in one plate and a second cell type in a second plate. This will avoid tying batch effects with cell types of interest. In addition, if more than one 384-well plate is needed for analysis, it is advisable to isolate cells in a single session, rather than spreading the isolation step over several days, if possible.
Lysis of both single cells and carrier/reference takes place in water through a freeze-heat cycle23. It is anticipated that most proteases have been denatured during this step. Trypsin is then added immediately following the denaturation step at a high concentration, many orders of magnitude greater in concentration than even the most abundant cellular proteases. By mass action, most products of protease activity will be due to trypsin.
Reduction/alkylation of cysteine residues is not performed in this protocol before trypsin digestion. Cysteine-containing peptides represent roughly 10% of tryptic peptides from the human proteome. We observe fewer cysteine-containing peptides using an approach without reduction/alkylation. These steps use reagents that are incompatible with labeling by TMT (NHS-ester chemistry) immediately following digestion.
In this TMT labeling strategy, the carrier and references are labeled with 126 and 127N, respectively, while single-cell and control wells are labeled with 128C through 135N. The two labels after the reference, 127C and 128N, are not used due to isotopic cross contamination arising from the carrier and reference channels, which are clearly much more abundant in peptide material than the single cells. In total, there are 12 TMT channels per set that can be used for labeling single cells or control wells with TMTpro-16plex, or 14 TMT channels per set with TMTpro-18plex.
The critical steps in the protocol to perform single-cell proteomics measurements using this protocol include preparation of the carrier, single-cell isolation, lysis, digestion, barcode labeling, and proper mass spectrometry parameters. These steps have been outlined in this document and have been extensively detailed elsewhere17,21,25. Each step has a corresponding plot or set of plots in DO-MS that allow easy quality control. For example, in the case of successful creation of the carrier, plots depicting the number of peptides identified, the labeling efficiency, and the percentage of miscleavage rate allow verification of its successful preparation. Representative DO-MS reports are included in this publication and can be seen at http://scope2.slavovlab.net/ for the original data16. These DO-MS reports allow for assessing optimization of the method in directions not yet investigated by the authors, such as longer LC gradients, different digestion enzymes, or alternative chemical barcodes.
Currently, the serial analysis of peptides by data-dependent acquisition algorithms limits the number of peptides that can be analyzed in an LC run of a reasonable length. This is in part due to the longer fill times required for the successful acquisition of enough ions for reliable single-cell quantification21. An inherent limitation to using the carrier is the lack of direct assessment of digestion and labeling efficiency of the single cells. Currently, one solution to this limitation is performing quality control on a larger sample processed alongside the single cells.
We proposed a number of opportunities for improving single-cell proteomics, such as building mass spectrometers with multiple analyzers. The current method allows the quantification of thousands of proteins from single mammalian cells at a speed of approximately 100 cells per day with commercially available reagents and equipment. SCoPE2, the second generation of the SCoPE-MS approach, significantly improved upon its predecessor with respect to the number of cells analyzable per unit time, the number of proteins analyzable per unit time, the time needed for sample preparation, the accessibility of reagents and equipment, and the overall cost of preparation and analysis per single cell. Membrane-bound proteins are accessible using the freeze-heat lysis and protease digestion, as shown in comparison to urea lysis23. The method identifies many proteins from the top third of the proteome with respect to abundance16. If the modified proteoform is in the top third of the proteome, and the modified peptide is amenable to mass spectrometry analysis (ionizes well, has a mass difference from unmodified peptide), it will more likely be amenable to this protocol. This method may be fruitfully applied in biological systems where there is meaningful heterogeneity amongst cells, such as in differentiation, senescence, or immunological responses (i.e., phagocytosis).

Single-cell analysis is widely used to study the level of heterogeneity within biological systems that would otherwise be indiscernible by&#160;bulk measurements1,2,3. Such cellular diversity can have functional consequences that can further the understanding of integral biological processes, from cancer cell therapeutic resistance4,5,6 to cell-to-cell heterogeneity in diabetes7,8,9,10. Many investigations have focused on measuring nucleic acids in single cells, by measuring genetic or transcriptomic levels and have enabled the classification of cell types and states. Such progress in the nucleic acid space, however, cannot fill the knowledge gap of post-transcriptional regulation11, driving the need for similarly high-throughput protein measurements in single cells12,13,14,15.
We have developed SCoPE216,17 to address the demand for rigorous and robust single-cell proteomics of mammalian systems. It is a multiplexed method, allowing for increased throughput by labeling and analyzing many cells in parallel18, in contrast to label-free mthods that analyze one cell at at time19,20. The sample preparation methodology, mass spectrometry approach, and subsequent data analysis steps allow for accurate quantification of hundreds to thousands of proteins per single cell. This method makes mass spectrometry analysis of single cells possible through the isobaric carrier21, a small bulk sample of cells (usually 25-200) that is biologically similar to the single-cell population of interest. This carrier material is multiplexed in a set with a reference channel of the same material and single cells through the use of tandem mass tags (TMT labels). The carrier channel reduces sample loss to surface areas and provides the ion backbone fragments for successful peptide identification. The reference channel, which is prepared in bulk and subsequently diluted down to 5-10 cell equivalents for each single-cell set, helps control technical variability in the analysis. Specifically, the reference allows for the normalization of variability caused by LC-MS/MS-related effects, such as ion sampling and ionization efficiencies. Usually, the reference and the carrier channels are made from the same cell population.
This sample preparation protocol is a second-generation method building upon SCoPE-MS22 by multiple synergistic improvements. The improvements include a cell lysis method that avoids sample clean-up, which is a common step of MS proteomics preparations. It uses mPOP (minimal ProteOmic sample Preparation)23, which is a freeze-heat lysis method. This method enabled lysis of single cells in smaller volumes and in multiwell plates rather than in vials, facilitating a higher throughput rate and automation by thermal cyclers. Altogether, this method has lowered the cost per single cell and increased quantitative accuracy as compared to SCoPE-MS16,24.
This protocol describes how to prepare single cells for proteomic analysis, including how to prepare the carrier and reference channels using TMTpro 18-plex isobaric labels. Mammalian cells that are in single-cell suspension and that can be isolated into 384-well plates are likely amenable to this protocol. Also included are representative results of successful single-cell sets, displaying several quality control plots generated by a R Shiny app, DO-MS25. Other published software tools26,27 and experimental guidelines17,21 have improved the adoption of this protocol. We hope this visual guide will further help researchers perform single-cell proteomics experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>384-well plate, standard PCR plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB1384</td>
			<td>Polypropylene plates should be used, if need to substitute. If substituted, levels of potential polymer contamination from other plates should be assessed prior to SCoPE2 preparation.</td>
		</tr>
		<tr>
			<td>Acetonitrile (for preparation of buffers), Optima LC-MS/MS grade</td>
			<td>Fisher Scientific</td>
			<td>A955-1</td>
			<td>This acetonitrile is used for any buffers, namely the 50% acetonitrile that is used to pass through wells after passing through the carrier and reference when combining SCoPE2 sets. 
			Caution: Acetonitrile is a flammable liquid. It can irritate skin, eyes, respiratory tract, and central nervous system. Be sure to wear personal protective equipment. Handle under a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Acetonitrile (for preparation of TMT ), anhydrous, 99.8%</td>
			<td>Sigma Aldrich</td>
			<td>271004-100ML</td>
			<td>This acetonitrile is used to resuspend TMT labels at the manufacturer concentration. 
			Caution: Acetonitrile is a flammable liquid. It can irritate skin, eyes, respiratory tract, and central nervous system. Be sure to wear personal protective equipment. Handle under a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Adhesive PCR plate foils</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB0626</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler vial screw thread caps</td>
			<td>Thermo Fisher Scientific</td>
			<td>C5000-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler vial spinner (i.e. myFuge 5)</td>
			<td>MTC Bio</td>
			<td>C2595</td>
			<td>This model does not have any speed control. It is simply used to colelct liquid at the bottom of autosampler vials.</td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Sigma Aldrich</td>
			<td>E1014-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear glass screw thread vials, 9 mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>60180-509</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid, Pierce, LC-MS/MS grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>85178</td>
			<td>Caution: Formic acid is a flammable liquid. It can cause serious eye damage or skin burns. Be sure to wear personal protective equipment. Handle in a well-ventilated area.</td>
		</tr>
		<tr>
			<td>Glass autosampler inserts, 9 mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>C4010-630</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxylamine (HA), 50% wt/vol</td>
			<td>Sigma Aldrich</td>
			<td>467804-50ML</td>
			<td>Caution: Hydroxylamine can cause skin irritation. Be sure to wear personal protective equipment.</td>
		</tr>
		<tr>
			<td>Mantis microfluidic chips, low-volume 3PFE chips</td>
			<td>Formulatrix</td>
			<td>MCLVPR2</td>
			<td>These chips are meant to be used with the Mantis microfluidics liquid handler to dispense organic solutions. These can be used to dispense TMT labels.</td>
		</tr>
		<tr>
			<td>Mantis microfluidic chips, low-volume silicone chips</td>
			<td>Formulatrix</td>
			<td>MCLVS12</td>
			<td>These chips are meant to be used with the Mantis microfluidics liquid handler to dispense aqueous solutions. These cannot be used to dispense TMT labels.</td>
		</tr>
		<tr>
			<td>Mantis microfluidic liquid handler</td>
			<td>Formulatrix</td>
			<td></td>
			<td>Liquid handler can be substituted. However, it is important to check if the system is compatible with 100% acetonitrile (as in the case of TMT labels), and that the system does not introduce polymer contamination or other type of contamination into the samples.</td>
		</tr>
		<tr>
			<td>Mantis PCR plate adapter with wide conical pins for automated plate handling</td>
			<td>Formulatrix</td>
			<td>232400</td>
			<td></td>
		</tr>
		<tr>
			<td>MassPREP peptide mixture</td>
			<td>Waters</td>
			<td>186002337</td>
			<td>Mixture of nine nontryptic peptides</td>
		</tr>
		<tr>
			<td>PCR tube spinner (i.e., 16-place microcentrifuge for 0.2 mL tubes)</td>
			<td>USA Scientific</td>
			<td>2621-0016</td>
			<td>This model does not have any speed control. It is simply used to collect liquid at the bottom of tubes.</td>
		</tr>
		<tr>
			<td>PCR tubes: TempAssure 0.2 mL PCR 8-tube strips</td>
			<td>USA Scientific</td>
			<td>1402-3900</td>
			<td>If substituted, levels of potential polymer contamination from other plates should be assessed prior to SCoPE2 preparation.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), 10x, pH 7.4, RNase-free</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate spinner (i.e., PlateFuge microcentrifuge)</td>
			<td>Benchmark Scientific</td>
			<td>Model C2000</td>
			<td>This model does not have any speed control. It is simply used to collect liquid at the bottom of wells.</td>
		</tr>
		<tr>
			<td>Thermal Cycler (i.e., C1000 Touch with 384-well module)</td>
			<td>Biorad</td>
			<td>1851138</td>
			<td></td>
		</tr>
		<tr>
			<td>TMTpro 16plex Label Reagent Set, 1 x 5 mg</td>
			<td>Thermo Fisher Scientific</td>
			<td>A44520</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate (TEAB), 1 M, pH 8.5</td>
			<td>Sigma Aldrich</td>
			<td>T7408100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Gold Mass Spectrometry Grade</td>
			<td>Promega</td>
			<td>V5280</td>
			<td>This is trypsin is of very high purity. Other trypsin reagents can vary in their levels of purity. Level of purity is important for achieving positive SCoPE2 results. 
			 
			Caution: Trypsin can cause skin, respiratory, and eye irritation. Be sure to wear personal protective equipment. Handle under a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Vortex (Analog vortex mixer)</td>
			<td>VWR</td>
			<td>Model 58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath sonicator (i.e., 2.8 L ultrasonic cleaner with digital timer)</td>
			<td>VWR</td>
			<td>97043964</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, Optima LC-MS/MS grade</td>
			<td>Fisher Scientific</td>
			<td>W6-1</td>
			<td>All solutions that need to be diluted or made are recommended to be made with mass-spectrometry grade water. Any dilutions and/or master mixes should be made fresh. Contamination resulting from lower-grade water can negatively affect peptide detection and single-cell data quality .</td>
		</tr>
	</tbody>
</table>

single-cell proteomics preparation for mass spectrometry analysis using freeze-heat lysis and an isobaric carrier

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down to the level of a single cell. Experiments using this method have achieved quantifying over 3,000 proteins across 1,500 single mammalian cells (500-1,000 proteins per cell) in 10 days of mass spectrometer instrument time. SCoPE2 leverages a freeze-heat cycle for cell lysis, obviating the need for clean-up of single cells and consequently reducing sample losses, while expediting sample preparation and simplifying its automation. Additionally, the method uses an isobaric carrier, which aids protein identification and reduces sample losses.
This video protocol provides detailed guidance to enable the adoption of automated single-cell protein analysis using only equipment and reagents that are widely accessible. We demonstrate critical steps in the procedure of preparing single cells for proteomic analysis, from harvesting up to injection to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, viewers are guided through the principles of experimental design with the isobaric carrier, quality control for both isobaric carrier and single-cell preparations, and representative results with a discussion of limitations of the approach.

Positive results from the protocol entail verifying that a number of critical steps in the protocol have been successfully performed; these include the preparation of the carrier, single-cell isolation, lysis, digestion, barcode labeling, and MS parameter optimization. DO-MS plots allow the easy assessment of the completion of each critical step in the protocol. A successfully prepared carrier, which usually corresponds to 25 and 200 cells in quantity (per set),&#160;is well-digested and well-labeled. Plots from DO-MS allow quantifying the intensity of the sample (to estimate the quantity), the digestion efficiency (ideally &#60;25% miscleavages), and the labeling efficiency (must be &#62;99%). Most critically, carrier samples that are not completely labeled may permit the cross reaction with the barcodes intended for single cells and add a spurious signal to the quantification of single-cell peptides.
Negative control wells include all experimental reagents but lack a cell. This allows not only background noise to be assessed but also cell isolation efficiency to be determined by providing a representative signal of an empty well. The DO-MS report offers plots to assess the measured signal of the control well alongside the single-cell wells to determine the success of cell isolation and background noise. Additionally, the quality of quantitation can be assessed by using the SCoPE2 pipeline (available on GitHub: github.com/SlavovLab/SCoPE2) or the SCP Bioconductor package29.
Digestion and labeling efficiency of the single cells may not be directly assayed as with the carrier, but DO-MS again provides plots that allow the estimation of the digestion and labeling efficiency. By including a control of 100-200 cells alongside the preparation of the single cells (i.e., receiving all the same reagent additions), the digestion and labeling efficiencies can be assessed for that control, and assumed to be shared by the single cells prepared alongside. Poor digestion will be indicated by a high ratio of the intensity of miscleaved peptides to their completely cleaved counterparts (Figure 1).
Another factor to consider in the sets is the fraction of missing data and the median reporter ion intensity in each TMT channel (Figure 2). When single cells are successfully prepared, the amount of missing data per cell and positive control is much lower than in negative control samples. Likewise, the median intensity of precursors is much higher in single cells and positive controls than for negative controls. The optimization of MS parameters has been discussed extensively elsewhere21, and optimal parameters can be determined using such tools as DO-MS or SCP Companion25,27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63802/63802fig01.jpg" /> 
Figure 1: Carrier preparation quality control. DO-MS plots depicting (A) labeling efficiency of a successfully prepared carrier at sites of labeling with TMT: the primary amine on the side chain of lysine and the peptide N-terminus, and (B) the digestion efficiency at amino acid residues of tryptic cleavage. Abbreviation: TMT = tandem mass tag. <a href="https://www.jove.com/files/ftp_upload/63802/63802fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63802/63802fig02.jpg" /> 
Figure 2: Single-cell preparation quality control. DO-MS plots depicting (A) the fraction of missing data in single cells (C5-10), controls (C13,16), carrier (C1), and reference (C2), and (B) the intensity of single cells and controls. Tags 127C, 128N, 131C, 132N, 133N, and 133C, which correspond to C3, C4, C11, C12, and C15, are unused in this particular set. The positive control consists of cellular material processed to peptides in bulk, then diluted to a level of 2-5 cells/&#956;L prior to labeling. The negative control consists of a well subjected to all preparatory steps used for single cells, just without the addition of a single cell. Abbreviation: TMT = tandem mass tag. <a href="https://www.jove.com/files/ftp_upload/63802/63802fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier at JoVE.com

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

In this protocol, we describe how to prepare mammalian cells for single-cell proteomics analysis via mass spectrometry using commercially available reagents and equipment, with options for both manual and automatic pipetting.

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the ...

single-cell-proteomics-preparation-for-mass-spectrometry-analysis

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3.7K Views.  Northeastern University. In this protocol, we describe how to prepare mammalian cells for single-cell proteomics analysis via mass spectrometry using commercially available reagents and equipment, with options for both manual and automatic pipetting.

Video: Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.