Lipopolysaccharide Regulates the Macrophage RNA-Binding Proteome.

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins' (RBPs') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs. Global RNA sequencing and LFQ proteomics were used to characterize the correlation of transcript and protein abundance changes in response to LPS at different time points with changes in protein-RNA binding. Il1α, MARCKS, and ACOD1 were noted as RBP candidates involved in innate immune signaling. The binding sites of the RBP and RNA conjugates at amino acid resolution were investigated by digesting the cross-linked oligonucleotide from peptides remaining after elution using Nuclease P1. The combined data sets provide directions for further studies of innate immune signaling regulation by RBP interactions with different classes of RNA.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) regulates toll-like receptor 4 signaling in macrophages.

MARCKS (myristoylated alanine-rich C-kinase substrate) is a membrane-associated protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (toll-like receptor 4).The presence of MARCKS and the formation of phospho-MARCKS in various cell types have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. We investigated the role of MARCKS in inflammation. Confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation.CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated the production of TNF and IL6, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes including innate immune response, inflammatory response, cytokine production, and molecular functions such as extracellularly ATP-gated cation channel activity, electron transfer activity and oxidoreductase activity, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory diseases.

Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) regulates Toll-like receptor 4 signaling in macrophages.

MARCKS (Myristoylated Alanine-rich C-kinase Substrate) is a membrane protein expressed in many cell types, including macrophages. MARCKS is functionally implicated in cell adhesion, phagocytosis, and inflammation. LPS (lipopolysaccharide) triggers inflammation via TLR4 (Toll-like receptor 4). The presence of MARCKS and the formation of phospho-MARCKS in macrophages have been described, but the role(s) of MARCKS in regulating macrophage functions remain unclear. To investigate the role of MARCKS during inflammation, we activated macrophages using LPS with or without the addition of a PKC inhibitor. We found that PKC inhibition substantially decreased macrophage IL6 and TNF cytokine production. In addition, confocal microscopy revealed that MARCKS and phospho-MARCKS increased localization to endosomes and the Golgi apparatus upon LPS stimulation. CRISPR-CAS9 mediated knockout of MARCKS in macrophages downregulated TNF and IL6 production, suggesting a role for MARCKS in inflammatory responses. Our comprehensive proteomics analysis together with real-time metabolic assays comparing LPS-stimulation of WT and MARCKS knock-out macrophages provided insights into the involvement of MARCKS in specific biological processes and signaling pathways, uncovering specific proteins involved in regulating MARCKS activity upon LPS stimulation. MARCKS appears to be a key regulator of inflammation whose inhibition might be beneficial for therapeutic intervention in inflammatory related diseases.

The minichromosome maintenance complex drives esophageal basal zone hyperplasia.

Eosinophilic esophagitis (EoE) is a chronic gastrointestinal disorder characterized by food antigen-driven eosinophilic inflammation and hyperproliferation of esophageal mucosa. By utilizing a large-scale, proteomic screen of esophageal biopsies, we aimed to uncover molecular drivers of the disease. Proteomic analysis by liquid chromatography-tandem mass spectrometry identified 402 differentially expressed proteins (DEPs) that correlated with the EoE transcriptome. Immune cell-related proteins were among the most highly upregulated DEPs in EoE compared with controls, whereas proteins linked to epithelial differentiation were primarily downregulated. Notably, in the inflamed esophageal tissue, all 6 subunits of the minichromosome maintenance (MCM) complex, a DNA helicase essential for genomic DNA replication, were significantly upregulated at the gene and protein levels. Furthermore, treating esophageal epithelial cells with a known inhibitor of the MCM complex (ciprofloxacin) blocked esophageal epithelial proliferation. In a murine model of EoE driven by overexpression of IL-13, ciprofloxacin treatment decreased basal zone thickness and reduced dilated intercellular spaces by blocking the transition of epithelial cells through the S-phase of the cell cycle. Collectively, a broad-spectrum proteomic screen has identified the involvement of the MCM complex in EoE and has highlighted MCM inhibitors as potential therapeutic agents for the disease.

EnsMOD: A Software Program for Omics Sample Outlier Detection.

Detection of omics sample outliers is important for preventing erroneous biological conclusions, developing robust experimental protocols, and discovering rare biological states. Two recent publications describe robust algorithms for detecting transcriptomic sample outliers, but neither algorithm had been incorporated into a software tool for scientists. Here we describe Ensemble Methods for Outlier Detection (EnsMOD) which incorporates both algorithms. EnsMOD calculates how closely the quantitation variation follows a normal distribution, plots the density curves of each sample to visualize anomalies, performs hierarchical cluster analyses to calculate how closely the samples cluster with each other, and performs robust principal component analyses to statistically test if any sample is an outlier. The probabilistic threshold parameters can be easily adjusted to tighten or loosen the outlier detection stringency. EnsMOD can be used to analyze any omics dataset with normally distributed variance. Here it was used to analyze a simulated proteomics dataset, a multiomic (proteome and transcriptome) dataset, a single-cell proteomics dataset, and a phosphoproteomics dataset. EnsMOD successfully identified all of the simulated outliers, and subsequent removal of a detected outlier improved data quality for downstream statistical analyses.

STAT4 Phosphorylation of T-helper Cells predicts surgical outcomes in Refractory Chronic Rhinosinusitis.

Objective: Chronic rhinosinusitis (CRS) impacts an estimated 5% to 15% of people worldwide, incurring significant economic healthcare burden. There is a urgent need for the discovery of predictive biomarkers to improve treatment strategies and outcomes for CRS patients. Study design: Cohort study of CRS patients and healthy controls using blood samples. Setting: Out-patient clinics. Methods: Whole blood samples were collected for flow cytometric analysis. Mechanistic studies involved the transfection of human primary T cells and Jurkat cells. Results: Our analysis began with a 63-69 year-old female patient diagnosed with refractory CRS,. Despite undergoing multiple surgeries, she continually faced sinus infections. Whole exome sequencing pinpointed a heterozygous IL-12Rb1 mutation situated in the linker region adjacent to the cytokine binding domain. When subjected to IL-12 stimulation, the patient's CD4 T-cells exhibited diminished STAT4 phosphorylation. However, computer modeling or T-cell lines harboring the same IL-12 receptor mutation did not corroborate the hypothesis that IL-12Rb could be responsible for the reduced phosphorylation of STAT4 by IL-12 stimulation. Upon expanding our investigation to a broader CRS patient group using the pSTAT4 assay, we discerned a subset of refractory CRS patients with abnormally low STAT4 phosphorylation. The deficiency showed improvement both in-vitro and in-vivo after exposure to Latilactobacillus sakei (aka Lactobacillus sakei), an effect at least partially dependent on IL-12. Conclusion: In refractory CRS patients, an identified STAT4 defect correlates with poor clinical outcomes after sinus surgery, which can be therapeutically targeted by Latilactobacillus sakei treatment. Prospective double-blind placebo-controlled trials are needed to validate our findings.

Absolute protein quantitation of the mouse macrophage Toll-like receptor and chemotaxis pathways.

The Toll-like receptor (TLR) and chemotaxis pathways are key components of the innate immune system. Subtle variation in the concentration, timing, and molecular structure of the ligands are known to affect downstream signaling and the resulting immune response. Computational modeling and simulation at the molecular interaction level can be used to study complex biological pathways, but such simulations require protein concentration values as model parameters. Here we report the development and application of targeted mass spectrometry assays to measure the absolute abundance of proteins of the mouse macrophage Toll-like receptor 4 (TLR4) and chemotaxis pathways. Two peptides per protein were quantified, if possible. The protein abundance values ranged from 1,332 to 227,000,000 copies per cell. They moderately correlated with transcript abundance values from a previously published mouse macrophage RNA-seq dataset, and these two datasets were combined to make proteome-wide abundance estimates. The datasets produced during this investigation can be used for pathway modeling and simulation, as well as for other studies of the TLR and chemotaxis pathways.

Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12+ macrophages.

Microbiota contribute to the induction of type 2 diabetes by high-fat/high-sugar (HFHS) diet, but which organs/pathways are impacted by microbiota remain unknown. Using multiorgan network and transkingdom analyses, we found that microbiota-dependent impairment of OXPHOS/mitochondria in white adipose tissue (WAT) plays a primary role in regulating systemic glucose metabolism. The follow-up analysis established that Mmp12+ macrophages link microbiota-dependent inflammation and OXPHOS damage in WAT. Moreover, the molecular signature of Mmp12+ macrophages in WAT was associated with insulin resistance in obese patients. Next, we tested the functional effects of MMP12 and found that Mmp12 genetic deficiency or MMP12 inhibition improved glucose metabolism in conventional, but not in germ-free mice. MMP12 treatment induced insulin resistance in adipocytes. TLR2-ligands present in Oscillibacter valericigenes bacteria, which are expanded by HFHS, induce Mmp12 in WAT macrophages in a MYD88-ATF3-dependent manner. Thus, HFHS induces Mmp12+ macrophages and MMP12, representing a microbiota-dependent bridge between inflammation and mitochondrial damage in WAT and causing insulin resistance.

Species-Specific Endotoxin Stimulus Determines Toll-Like Receptor 4- and Caspase 11-Mediated Pathway Activation Characteristics.

The innate immune system is the body's first line of defense against pathogens and its protection against infectious diseases. On the surface of host myeloid cells, Toll-like receptor 4 (TLR4) senses lipopolysaccharide (LPS), the major outer membrane component of Gram-negative bacteria. Intracellularly, LPS is recognized by caspase 11 through the noncanonical inflammasome to induce pyroptosis-an inflammatory form of lytic cell death. While TLR4-mediated signaling perturbations result in secretion of cytokines and chemokines that help clear infection and facilitate adaptive immunity, caspase 11-mediated pyroptosis leads to the release of damage-associated molecular patterns and inflammatory mediators. Although the core signaling events and many associated proteins in the TLR4 signaling pathway are known, the complex signaling events and protein networks within the noncanonical inflammasome pathway remain obscure. Moreover, there is mounting evidence for pathogen-specific innate immune tuning. We characterized the major LPS structures from two different pathogens, modeled their binding to the surface receptors, systematically examined macrophage inflammatory responses to these LPS molecules, and surveyed the temporal differences in global protein secretion resulting from TLR4 and caspase 11 activation in macrophages using mass spectrometry (MS)-based quantitative proteomics. This integrated strategy, spanning functional activity assays, top-down structural elucidation of endotoxins, and secretome analysis of stimulated macrophages, allowed us to identify crucial differences in TLR4- and caspase 11-mediated protein secretion in response to two Gram-negative bacterial endotoxins. IMPORTANCE Macrophages and monocytes are innate immune cells playing an important role in orchestrating the initial innate immune response to bacterial infection and the tissue damage. This response is facilitated by specific receptors on the cell surface and intracellularly. One of the bacterial molecules recognized is a Gram-negative bacteria cell wall component, lipopolysaccharide (LPS). The structure of LPS differs between different species. We have characterized the innate immune responses to the LPS molecules from two bacteria, Escherichia coli and Bordetella pertussis, administered either extracellularly or intracellularly, whose structures we first determined. We observed marked differences in the temporal dynamics and amounts of proteins secreted by the innate immune cells stimulated by any of these molecules and routes. This suggests that there is specificity in the first line of response to different Gram-negative bacteria that can be explored to tailor specific therapeutic interventions.

Molecular Mechanisms of the Toll-Like Receptor, STING, MAVS, Inflammasome, and Interferon Pathways.

Pattern recognition receptors (PRRs) form the front line of defense against pathogens. Many of the molecular mechanisms that facilitate PRR signaling have been characterized in detail, which is critical for the development of accurate PRR pathway models at the molecular interaction level. These models could support the development of therapeutics for numerous diseases, including sepsis and COVID-19. This review describes the molecular mechanisms of the principal signaling interactions of the Toll-like receptor, STING, MAVS, and inflammasome pathways. A detailed molecular mechanism network is included as Data Set S1 in the supplemental material.

LPS Tolerance Inhibits Cellular Respiration and Induces Global Changes in the Macrophage Secretome.

Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.

Proteomic profile of vitreous in patients with tubercular uveitis.

OBJECTIVE: To elucidate disease-specific host protein profile in vitreous fluid of patients with intraocular inflammation due to tubercular uveitis (TBU). METHODS: Vitreous samples from 13 patients with TBU (group A), 7 with non-TBU (group B) and 9 with no uveitis (group C) were analysed by shotgun proteomics using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). Differentially expressed proteins (DEPs) were subjected to pathway analysis using WEB-based Gene SeT Analysis Toolkit software. RESULTS: Compared to control groups (B + C combined), group A (TBU) displayed 32 (11 upregulated, 21 downregulated) DEPs, which revealed an upregulation of coagulation cascades, complement and classic pathways, and downregulation of metabolism of carbohydrates, gluconeogenesis, glucose metabolism and glycolysis/gluconeogenesis pathways. When compared to group B (non-TBU) alone, TBU displayed 58 DEPs (21 upregulated, 37 downregulated), with an upregulation of apoptosis, KRAS signaling, diabetes pathways, classic pathways, and downregulation of MTORC1 signaling, glycolysis/gluconeogenesis, and glucose metabolism. CONCLUSION: This differential protein profile provides novel insights into the molecular mechanisms of TBU and a baseline to explore vitreous biomarkers to differentiate TBU from non-TBU, warranting future studies to identify and validate them as a diagnostic tool in TBU. The enriched pathways generate interesting hypotheses and drive further research.

Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages.

We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identified by weighted correlation network analysis (WGCNA) based on their correlated movement into or out of SEC fractions following stimulation, presenting an analysis method for SEC-MS that does not rely on established databases. We highlight two modules of interest: one linked to the apoptosis signal-regulating kinase (ASK) signalosome and the other containing poly(ADP-ribose) polymerase 9 (PARP9). Finally, PARP inhibition was used to perturb the characterized systems, demonstrating the importance of ADP-ribosylation for the global interactome. All post-translational modification (PTM) and interactome data have been aggregated into a meta-database of 6729 proteins, with ADP-ribosylation characterized on 2905 proteins and phosphorylation characterized on 2669 proteins. This database-titled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation, and Complex Dynamics-serves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.

Multi-Omics Strategies Uncover Host-Pathogen Interactions.

With the success of the Human Genome Project, large-scale systemic projects became a reality that enabled rapid development of the systems biology field. Systems biology approaches to host-pathogen interactions have been instrumental in the discovery of some specifics of Gram-negative bacterial recognition, host signal transduction, and immune tolerance. However, further research, particularly using multi-omics approaches, is essential to untangle the genetic, immunologic, (post)transcriptional, (post)translational, and metabolic mechanisms underlying progression from infection to clearance of microbes. The key to understanding host-pathogen interactions lies in acquiring, analyzing, and modeling multimodal data obtained through integrative multi-omics experiments. In this article, we will discuss how multi-omics analyses are adding to our understanding of the molecular basis of host-pathogen interactions and systemic maladaptive immune response of the host to microbes and microbial products.

Host-pathogen dynamics through targeted secretome analysis of stimulated macrophages.

The pattern recognition receptors (PRRs) facilitate an organism's first line of defense against interlopers and shape the overall innate immune response through sensing and sampling pathogen-associated molecular patterns (PAMPs). The Toll-like receptor (TLR) family is the prototypic PRR family. Upon recognition of PAMPs, TLRs promote MyD88 dependent and independent responses. Understanding how different PAMPs are recognized by their specific TLRs and how pathogen recognition initiates immune activation is an intense area of research. Previously, we have reported the discovery of the temporal changes in signaling cascades of macrophage proteome and secretome post-stimulation with three different PAMPs. To extend our global proteomics approach to targeted protein abundance quantification, we describe the macrophage secretome targeted proteomics assay. We chose three different pathogens that specifically stimulate diverse TLRs (TLR2, TLR4, and TLR7). Using a simple targeted proteomics approach, combining data-dependent acquisition with an inclusion list, an array of cytokines, chemokines, and transcription factors can be profiled for their secretome abundance. This strategy facilitates the profiling and validation of pathogen-specific temporal changes in the macrophage secretome.

Application of targeted mass spectrometry in bottom-up proteomics for systems biology research.

The enormous diversity of proteoforms produces tremendous complexity within cellular proteomes, facilitates intricate networks of molecular interactions, and constitutes a formidable analytical challenge for biomedical researchers. Currently, quantitative whole-proteome profiling often relies on non-targeted liquid chromatography-mass spectrometry (LC-MS), which samples proteoforms broadly, but can suffer from lower accuracy, sensitivity, and reproducibility compared with targeted LC-MS. Recent advances in bottom-up proteomics using targeted LC-MS have enabled previously unachievable identification and quantification of target proteins and posttranslational modifications within complex samples. Consequently, targeted LC-MS is rapidly advancing biomedical research, especially systems biology research in diverse areas that include proteogenomics, interactomics, kinomics, and biological pathway modeling. With the recent development of targeted LC-MS assays for nearly the entire human proteome, targeted LC-MS is positioned to enable quantitative proteomic profiling of unprecedented quality and accessibility to support fundamental and clinical research. Here we review recent applications of bottom-up proteomics using targeted LC-MS for systems biology research. SIGNIFICANCE: Advances in targeted proteomics are rapidly advancing systems biology research. Recent applications include systems-level investigations focused on posttranslational modifications (such as phosphoproteomics), protein conformation, protein-protein interaction, kinomics, proteogenomics, and metabolic and signaling pathways. Notably, absolute quantification of metabolic and signaling pathway proteins has enabled accurate pathway modeling and engineering. Integration of targeted proteomics with other technologies, such as RNA-seq, has facilitated diverse research such as the identification of hundreds of "missing" human proteins (genes and transcripts that appear to encode proteins but direct experimental evidence was lacking).

Multi-omics Comparative Analysis Reveals Multiple Layers of Host Signaling Pathway Regulation by the Gut Microbiota.

The bodies of mammals are hosts to vast microbial communities composed of trillions of bacteria from thousands of species, whose effects on health and development have begun to be appreciated only recently. In this investigation, an integrated analysis combining proteomics and transcriptomics was used to quantitatively compare the terminal ilia from conventional and germfree mice. Female and male mice responded similarly to the microbiota, but C57BL/10A mice responded more strongly than BALB/c mice at both the transcriptome and proteome levels. The microbiota primarily caused upregulation of immunological pathways and downregulation of metabolic pathways in the conventional mice. Many of the affected pathways were altered only at either the transcriptome or proteome level. Of the pathways that were affected at both levels, most were affected concordantly. The discordant pathways were not principally involved in the immune system but instead were related to metabolism, oxidative phosphorylation, protein translation, transport, and turnover. To broaden the discovery of affected host pathways, a meta-analysis was performed using intestinal transcriptomics data from previously published studies of germfree versus conventional mice with diverse microbiota populations. Similar transcript-level responses to the microbiota were found, and many additional affected host pathways were discovered. IMPORTANCE Multiple host pathways were affected by its adaptation to the microbiota. We have found significant transcriptome-proteome discordance caused by the microbiota. This discovery leads to the definite conclusion that transcript-level analysis is not sufficient to predict protein levels and their influence on the function of many specific cellular pathways, so only analysis of combinations of the quantitative data determined at different levels will lead to a complete understanding of the complex relationships between the host and the microbiota. Therefore, our results demonstrate the importance of using an integrative approach to study host-microbiota interaction at the molecular level.

Proteome and Secretome Analysis Reveals Differential Post-transcriptional Regulation of Toll-like Receptor Responses.

The innate immune system is the organism's first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level.

The development of SRM assays is transforming proteomics research.

Bottom-up targeted proteomics using SRM is a powerful analytical technology, but it requires the development of SRM assays, which is a complex procedure. Whereas proteome-wide SRM assays have recently been developed for a small number of species, this is not so for the mouse. In this issue, Percy et al. report the development of hundreds of mouse SRM assays. Their development required shotgun MS to identify proteotypic peptides, synthesis, and LC-MS characterization of peptide standards, and interlaboratory SRM to robustly assess the quality of the assays. The resulting SRM assays are intended to be used to analyze mouse plasma and cardiac tissue, primarily for cardiovascular disease and cancer research.

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 α-subunit) is described in detail.