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Additional file 2. Review history.

The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF's commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways, but their transient nature and low abundance makes their analysis challenging. Here we follow up on our sPRG heavy-labeled phosphopeptide standard study designed to address issues encountered in phosphopeptide experiments. This standard is constructed of 150 heavy-labeled phosphopeptides spanning seven different signaling pathways and will be useful to test the effectiveness of phosphopeptide enrichment workflows, as an internal instrument and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. In our initial characterization and validation of this standard (sPRG 2018-2019 study), we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to participants with optimized and standardized enrichment and acquisition methods. Data independent acquisition (DIA) was performed on 8 gas phase fractionated injections. As an extension of this initial characterization and to prepare for a larger biological study, we present progress towards a universal single inject targeted data acquisition method using our standard which aims to be accurate, sensitive, scalable, and easily implemented and executed without the need for retention time scheduling. Two approaches were taken that both target the heavy isotope labeled internal standard peptides and trigger a mass offset scan of the endogenous light. The first method, QuanDirect, monitors for heavy y1 fragments and can be implemented on tribrid instrument platforms. The second method, SureQuant, monitors for select fragment ions and can be implemented on both tribrid and hybrid instruments.

With the advent of more sensitive and higher duty cycle mass spectrometers, analysis of limited samples, e.g., limited cell populations, microneedle biopsies, extracellular vesicles isolated from minute volumes of physiological fluids (i.e., liquid biopsies) or even single cells, is getting more feasible. However, each step of the analytical workflow, including sample preparation, liquid chromatography (LC) separation, interfacing with mass spectrometry (MS), MS data acquisition, data analysis and data interpretation requires additional advancements to enable deep proteomic profiling of such limited samples. In this study, we investigated MS data acquisition parameters on two different, commonly used mass spectrometers to enhance the sensitivity of proteomic profiling of limited samples. Our experiments indicate that nanoLC-MS/MS-based proteomic analysis of scarce samples requires adjustments of the commonly used default data acquisition settings on a mass spectrometer that work effectively for conventional sample amounts. Here we show that by using specific values of the ion injection time and AGC minimum settings in MS/MS data acquisition depending on the amount of sample results in an increase in the numbers of identified peptides and proteins. We applied commonly used settings, and then altered them to analyze HeLa tryptic digest at the levels of 1, 10 and 100 ng, which are equivalent to approximately 5, 50 and 500 cells on either an Orbitrap QE HFX or a Lumos Fusion (Thermo Fisher Scientific). By changing certain parameters for each sample amount, we were able to identify 2-4-fold more peptides and proteins, and therefore, significantly improve the protein and peptide profiling efficiency, in comparison to the commonly used settings. However, this is only a small step toward ultra-sensitive proteomic profiling. Improvements in LC separation and column material as well as sample preparation are also critical for further advances in highly sensitive analysis.

The fate and physiology of individual cells are controlled by protein interactions. We recently developed SCoPE-MS, a method for direct analysis of proteomes of single cells by LC-MS/MS. SCoPE-MS is enabled by isobaric-tag multiplexing of peptides from single cells together with carrier material, which serves both to minimize sample losses to equipment surfaces and enhance peptide identifications. Yet, our ability to quantitatively analyze proteins in single cells has remained limited. To overcome this barrier, we developed SCoPE2. SCoPE2 lowers cost and hands-on time by introducing automated and miniaturized sample preparation while increasing quantitative accuracy using only commercially available equipment and reagents. Additionally, SCoPE2 accomplishes increased sample preparation throughput and increased measurement throughput. Using SCoPE2, we quantified over 2,700 proteins in 1,018 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allowed us to discern single cells by cell type. Parallel measurements of transcripts by 10x Genomics scRNA-seq indicate that most genes had similar responses at the protein and RNA levels, though the responses of hundreds of genes differed. In this talk, we present users with how to begin adopting SCoPE2 in their lab. Executing a SCoPE2 experiment has similarities to conventional proteomics sample preparation as well as pitfalls and deviations unique to working with single cells. We discuss successful strategies, including study design and reagent selection, how to avoid common pitfalls, and the range of common equipment that can be used to execute SCoPE2 sample prep. Additionally, we discuss approaches to optimizing LC-MS/MS instrumentation for SCoPE2 samples. We hope to give users ideas about how to successfully design and execute a SCoPE2 experiment to facilitate the broad adoption of automated and quantitative single-cell analysis of proteins by mass-spectrome.

The mission of the ABRF Proteomics Standards Research Group (sPRG) is to identify and implement technical standards that reflect the ABRF's commitment to accuracy, clarity, and consistency in the field of proteomics. There is broad interest in quantifying protein phosphorylation alterations in cellular signaling pathways under different conditions. The transient nature and low abundance of many phosphorylation sites makes this analysis challenging. Here we report on the follow up of the two-year sPRG study designed to target various issues encountered in phosphopeptide experiments. We have constructed a pool of heavy-labeled phosphopeptides that will enable core facilities to rapidly develop assays. Our pool contains over 150 phosphopeptides that have been previously observed in mass spectrometry data sets. The specific peptides have been chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling pathways: AMPK, death and apoptosis, ErbB, insulin/IGF-1, mTOR, PI3K/AKT, and stress (p38/SAPK/JNK). We feel this pool will enable researchers to test the effectiveness of their enrichment workflows and to provide a benchmark for a cross lab study. This standard should be helpful in number of ways, including providing a complete workflow solution for phosphopeptide enrichment, as an internal enrichment and chromatography calibrant, and as a pre-built biological assay for a wide variety of signaling pathways. Previously, we mixed the standard into an activated HeLa tryptic digest and distributed the mixture to over 60 ABRF member and nonmember laboratories around the world. We asked participants to enrich phosphopeptides out of the HeLa background and report ratios of the heavy phosphopeptides to the endogenous levels. In the current study, we continue validation of the standard within various RG group/ABRF members' laboratories. The aim of this follow up study is to provide reagents, an optimized phosphopeptide enrichment protocol, instrument acquisition method parameters, and data analysis templates.

The fate and physiology of individual cells are controlled by protein interactions. Yet, our ability to quantitatively analyze proteins in single cells has remained limited. To overcome this barrier, we developed SCoPE2. It lowers cost and hands-on time by introducing automated and miniaturized sample preparation while substantially increasing quantitative accuracy. These advances enabled us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiated into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 2,700 proteins in 1,018 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allowed us to discern single cells by cell type. Furthermore, the data uncovered a continuous gradient of proteome states for the macrophage-like cells, suggesting that macrophage heterogeneity may emerge even in the absence of polarizing cytokines. Parallel measurements of transcripts by 10x Genomics scRNA-seq suggest that SCoPE2 samples 20-fold more copies per gene, thus supporting quantification with improved count statistics. Joint analysis of the data indicated that most genes had similar responses at the protein and RNA levels, though the responses of hundreds of genes differed. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass-spectrometry.

Western blotting method to detect thiophosphorylated Src. Src (catalytic domain, 295 nM) was incubated alone (unphos), with 1 mM ATP and 5 mM MgCl2 (phos), or with 1 mM ATPγS and 10 mM NiCl2 (thiophos) for 30 min at 30 °C. The reactions were analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody (top) and with anti-Src (pY416) antibody (bottom). In the right-hand lane of the gel, four times the amount of Src was loaded as in the other lanes. (PDF 466 kb)

The ABRF Proteomics Standards Research Group (sPRG) is reporting the progress of a two-year study (2012–2014) which focuses on the generation of interassay, interspecies, and interlaboratory peptide standard that can be used for normalization of protein abundance measurements in mass spectrometry based quantitative proteomics analyses. The standard has been formulated as two mixtures: 1,000 stable isotope 13C/15N-labeled (SIL) synthetic peptides alone, and peptides mixed with a tryptic digest of a HEK 293 cell lysate. The sequences of the synthetic peptides were derived from 552 proteins conserved across proteomes of commonly analyzed species: Homo sapiens, Mus musculus and Rattus norvegicus. The selected peptides represent a full range of hydrophobicities and isoelectric points, typical of tryptic peptides derived from complex proteomic samples. The standard was designed to represent proteins of various concentrations, spanning three orders of magnitude. First year efforts were focused on selection of appropriate protein and peptide candidates, peptide synthesis, quality assessment and LC-MS/MS evaluation conducted in laboratories of sPRG members. Using a variety of instrumental configurations and bioinformatics approaches, a thorough characterization of all 1,000 peptides was established. In the second year, the group launched the study to the entire proteomics community. A lyophilized mixture of HEK 293 tryptic digest cell lysate spiked with the 1,000 SIL peptide standards was provided to each participant. Also provided were a Skyline tutorial, tutorial datasets, three MS/MS spectral libraries generated from linear ion-trap (CID), Q-TOF/QQQ (CID), or Orbitrap (HCD) instrumentation, and a Panorama data repository. Participants were asked to analyze the sample in triplicate and calculate ratios of the spiked SIL to endogenous peptides and coefficients of variance for each peptide. Over 40 datasets were returned, and results following thorough characterization of the standard using various instrumental configurations will be reported.