Comprehensive Analysis Of Proteins Research Articles

PPI-GA: A Novel Clustering Algorithm to Identify Protein Complexes within Protein-Protein Interaction Networks Using Genetic Algorithm

Comprehensive analysis of proteins to evaluate their genetic diversity, study their differences, and respond to the tensions is the main subject of an interdisciplinary field of study called proteomics. The main objective of the proteomics is to detect and quantify proteins and study their post-translational modifications and interactions using protein chemistry, bioinformatics, and biology. Any disturbance in proteins interactive network can act as a source for biological disorders and various diseases such as Alzheimer and cancer. Most current computational methods for discovering protein complexes are usually based on specific topological characteristics of protein-protein networks (PPI). To identify the protein complexes, in this paper, we, first, present a new encoding method to represent solutions; we then propose a new clustering algorithm based on the genetic algorithm, named PPI-GA, employing a new multiobjective quality function. The proposed algorithm is evaluated on two gold standard and real-world datasets. The result achieved demonstrates that the proposed algorithm can detect important protein complexes, and it provides more accurate results compared with state-of-the-art protein complex identification algorithms.

Detailed Postmortem Profiling of Inflammatory Mediators Expression Revealed Post-inflammatory Alternation in the Superior Temporal Gyrus of Schizophrenia.

Recent studies have lent support to the possibility that inflammation is associated with the pathology of schizophrenia. In the study of measurement of inflammatory mediators, which are markers of inflammation, elevated inflammatory cytokine levels in the brain and blood have been reported in patients with schizophrenia. Several postmortem brain studies have also reported changes in the expression of inflammatory cytokines. However, it is not clear how these elevated inflammatory cytokines interact with other inflammatory mediators, and their association with the pathology of schizophrenia. We comprehensively investigated the expression of 30 inflammatory mediators in the superior temporal gyrus (STG) of 24 patients with schizophrenia and 26 controls using a multiplex method. Overall, inflammatory mediator expression in the STG was mostly unchanged. However, the expression of interleukin (IL)1-α and interferon-gamma-inducible protein (IP)-10 was decreased [IL-1α, median (IQR), 0.51 (0.37–0.70) vs. 0.87 (0.47–1.23), p = 0.01; IP-10, 13.99 (8.00–36.64) vs. 30.29 (10.23–134.73), p = 0.05], whereas that of IFN-α was increased [2.34 (1.84–4.48) vs. 1.94 (1.39–2.36), p = 0.04] in schizophrenia, although these alterations did not remain significant after multiple testing. Clustering based on inflammatory mediator expression pattern and analysis of upstream transcription factors using pathway analysis revealed that the suppression of IL-1α and IP-10 protein expression may be induced by regulation of a common upstream pathway. Neuroinflammation is important in understanding the biology of schizophrenia. While neuroimaging has been previously used, direct observation to determine the expression of inflammatory mediators is necessary. In this study, we identified protein changes, previously unreported, using comprehensive protein analysis in STG. These results provide insight into post-inflammatory alternation in chronic schizophrenia.

Comprehensive serum proteomic analysis in early endometrial cancer

ObjectiveEndometrial cancer is the most common gynecologic cancer and yet much is still unknown about this disease. Our goal was to identify unique biomarkers of disease by performing a comprehensive proteomic analysis of early stage, low-grade endometrial cancer through analysis of serum collected from patients pre- and post-definitive surgery. MethodsWe used mass spectrometry (MS)-based proteomics to identify serum proteins from these patients. Serum samples from women undergoing hysterectomy with bilateral salpingo-oophorectomy for benign reasons served as control samples for the correlative studies. We then correlated our findings with The Cancer Genome Atlas (TCGA) database for additional confirmation. ResultsThe Ingenuity Pathway Analysis of proteins that were differentially expressed in endometrial cancer showed increased cell survival and decreased organismal death, the most common hallmarks of cancer. We identified over expression of FAM83D (family with sequence similarity 83, member D) in the serum of patients with early stage low-grade endometrial cancer and verified the same in the endometrial cancer cell lines and patient tumors. We also confirmed our hypothesis that FAM83D may serve as a biomarker for endometrial cancer in a cohort of patients with endometrial cancer from The Cancer Genome Atlas (TCGA) project. ConclusionComprehensive proteomic analysis is a feasible strategy for potential biomarker identification. Using this technique, FAM83D was identified as a candidate biomarker in early endometrial cancer in our patient samples and was not present in benign control samples. FAM83D has been associated with poor clinical outcomes in several human malignancies. SignificanceOur manuscript describes an alternative approach to comprehensive protein analysis in a model pre and post tumor removal for a sample of patients with early endometrial cancer. The model is innovative and the findings of over expression FAM83D in this population of early cancer may be useful in the study of a disease where there are few biomarkers or targetable therapies.

Shotgun Proteomics and Mass Spectrometry as a Tool for Protein Identification and Profiling of Bio-Carrier-Based Therapeutics on Human Cancer Cells.

Shotgun proteomics has been widely applied to study proteins in complex biological samples. Combination of high-performance liquid chromatography with mass spectrometry has allowed for comprehensive protein analysis with high resolution, sensitivity, and mass accuracy. Prior to mass spectrometry analysis, proteins are extracted from biological samples and subjected to in-solution trypsin digestion. The digested proteins are subjected for clean-up and injected into the liquid chromatography-mass spectrometry system for peptide mass identification. Protein identification is performed by analyzing the mass spectrometry data on a protein search engine software such as PEAKS studio loaded with protein database for the species of interest. Results such as protein score, protein coverage, number of peptides, and unique peptides identified will be obtained and can be used to determine proteins identified with high confidence. This method can be applied to understand the proteomic changes or profile brought by bio-carrier-based therapeutics in vitro. In this chapter, we describe methods in which proteins can be extracted for proteomic analysis using a shotgun approach. The chapter outlines important in vitro techniques and data analysis that can be applied to investigate the proteome dynamics.

Optical imaging of single-protein size, charge, mobility, and binding

Detection and identification of proteins are typically achieved by analyzing protein size, charge, mobility and binding to antibodies, which are critical for biomedical research and disease diagnosis and treatment. Despite the importance, measuring these quantities with one technology and at the single-molecule level has not been possible. Here we tether a protein to a surface with a flexible polymer, drive it into oscillation with an electric field, and image the oscillation with a near field optical imaging method, from which we determine the size, charge, and mobility of the protein. We also measure antibody binding and conformation changes in the protein. The work demonstrates a capability for comprehensive protein analysis and precision protein biomarker detection at the single molecule level.

Shotgun Proteomics of Plant Plasma Membrane and Microdomain Proteins Using Nano-LC-MS/MS.

Shotgun proteomics allows for the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. However, this method is not fully applicable for highly hydrophobic proteins with multiple transmembrane domains. In order to solve this problem, we here describe a shotgun proteomics method using nano-LC-MS/MS for proteins in the plasma membrane and plasma membrane microdomain fractions. The results obtained are easily applicable to label-free protein semiquantification.

The human secretome.

The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immunoassays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.

Protein–ligand interaction-guided discovery of novel VEGFR-2 inhibitors

As an effective target in abnormal angiogenesis-related tumor treatment, VEGFR-2 has small-molecule inhibitors of various scaffolds being approved for treating diseases such as renal carcinoma, non-small cell lung cancer, etc. However, endogenous and acquired drug resistance are still considered to be the main contributors for the failure of VEGFR-2 clinical candidates. Therefore, development of novel VEGFR-2 inhibitors is still urgently needed in the market but also challenging. In this work, residues including Asp1046, Ile1025, HIS1026, Cys919 and Lys868 were identified as the most important residues for Hbonded interaction, while His1026, Asp1046, Glu885, Ile1025 and Leu840 exhibited critical role for the nonbonded interactions through a comprehensive analysis of protein–ligand interactions, which plays critical roles in the binding of compounds and targets. Guided by the analysis of binding interactions, a total of 10 novel VEGFR-2 inhibitors based on N-methyl-4-oxo-N-propyl-1,4-dihydroquinoline-2-carboxamide scaffold were discovered through fragment-based drug design and structure-based virtual screening, which expands the chemical space of current VEGFR-2 inhibitors. Biological activity evaluation showed that even though the enzymatic activity of these compounds against VEGFR-2 were inferior to that of the positive controls sorafenib and motesanib, compound I-10 showed moderate HepG2 cell inhibitory activity with an IC50 value of 33.65 μM and eight compounds exhibited moderate or higher HUVEC inhibitory activity in the range of 19.54–57.98 μM compared to the controls. Particularly, the HUVEC inhibitory activity of compound I-6 (IC50 = 19.54 μM) outperformed motesanib and can be used as starting points for further optimization and development for cancer treatment.Communicated by Ramaswamy H. Sarma

Exploration of Proteins Involved in Acquisition of Resistance to Cetuximab

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (Mabs) show high efficacy in about 50% of colorectal cancer (CRC) patients with wild-type KRAS. However, < 20% of patients with KRAS wild-type CRC have continued therapeutic effects with these agents, and acquired resistance to treatment has become a serious clinical problem. In this study, to clarify the factors related to acquisition of resistance to cetuximab (Cmab) and establish countermeasures against such acquired resistance, we conducted a comprehensive protein analysis via a proteomics approach using acquired resistance cell lines derived from Cmab-sensitive CRC cell lines and original cell lines. Cmab-acquired resistance cell lines were generated by continuous exposure of SW48 and C99 cell lines to Cmab. Expression of dCK and zinc finger and BTB domain-containing protein 41 (ZBTB41) increased more than 10-fold, and dual specificity protein phosphatase 3 (DUS3) expression decreased by less than 1/10 with acquisition of resistance to Cmab in both C99 and SW48 cell lines. Because overexpression of dCK is known as a positive indicator of efficacy of nucleoside analogs such as cytarabine or gemcitabine, it is considered that nucleoside analogs activated by dCK may be useful agents in treatment of cancers with acquired Cmab-resistance. In the future, we need to clarify the usefulness of these drugs for the treatment of Cmab resistant CRC and to assess the possibility of restoration of Cmab sensitivity by regulation of ZBTB41 and DUS3 expression.Keyword : cetuximab, colorectal cancer, acquired resistance, protein, dCK, ZBTB41

Integrated Analysis of Transcriptomic and Proteomics Data Reveals the Induction Effects of Rotenoid Biosynthesis of Mirabilis himalaica Caused by UV-B Radiation.

Mirabilis himalaica (Edgew.) Heimerl is one of the most important genuine medicinal plants in Tibet, in which the special plateau habitat has been associated with its excellent medicinal quality and efficacy. However, the mechanisms by which environmental factors affect biosynthesis of secondary metabolic components remain unclear in this species. In this study, RNA sequencing and iTRAQ (isobaric Tags for Relative and Absolute Quantification) techniques were used to investigate the critical molecular “events” of rotenoid biosynthesis responding to UV-B radiation, a typical plateau ecological factor presented in native environment-grown M. himalaica plants. A total of 3641 differentially expressed genes (DEGs) and 106 differentially expressed proteins (DEPs) were identified in M. himalaica between UV-B treatment and control check (CK). Comprehensive analysis of protein and transcript data sets resulted in 14 and 7 DEGs from the plant hormone signal transduction and phosphatidylinositol signaling system pathways, respectively, being significantly enriched. The result showed that the plant hormone signal transduction and phosphatidylinositol signaling system might be the key metabolic strategy of UV-B radiation to improve the biosynthesis of rotenoid in M. himalaica. At same time, most of the DEGs were associated with auxin and calcium signaling, inferring that they might drive the downstream transmission of these signal transduction pathways. Regarding those pathways, two chalcone synthase enzymes, which play key roles in the biosynthesis of rotenoid that were thought as the representative medicinal component of M. himalaica, were significantly upregulated in UV-B radiation. This study provides a theoretical basis for further exploration of the adaptation mechanism of M. himalaica to UV-B radiation, and references for cultivation standardization.

Dose-Dependent Effects of FKRP Gene-Replacement Therapy on Functional Rescue and Longevity in Dystrophic Mice

Muscular dystrophy-dystroglycanopathies (MDDGs) resulting from fukutin-related protein (FKRP) gene mutations are rare disorders that result in a wide spectrum of clinical severity based on the age of onset, the degree of myogenic atrophy, and/or neurologic involvement. There is no cure for any of the FKRP-related disorders, and few options are available for symptom management. Herein, we examine the longitudinal effects of a dose-escalation study to evaluate the safety and therapeutic potential of FKRP gene-replacement therapy in a p.P448L (FKRPP448L) mouse model of MDDG. A recombinant adeno-associated virus (AAV) serotype 9 vector expressing human FKRP (AAV9-FKRP) was systemically administered to FKRPP448L mice at 5 weeks of age, when early onset of the disease is evidenced. A comprehensive analysis of protein and gene expression, histopathology, skeletal muscle function, and cardiorespiratory function was performed over short (9-week) and/or long-term (52-week) study periods. Additional studies assessed the impact of FKRP gene-replacement therapy on lifespan at an advanced stage of disease progression. Results indicate that treatment intervention can restore the biochemical defects in a dose-dependent manner, with potential for improvement in the trajectory of disease progression and extension of the expected lifespan. This study supports the initiation of early-stage clinical trials for FKRP-related disorders.

A new memetic algorithm for multiple graph alignment

A new memetic algorithm for multiple graph alignment

Comprehensive protein analysis of the transport system in a connective tissue of the inner ear

Comprehensive protein analysis of the transport system in a connective tissue of the inner ear

A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core

The MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) is a free, one-stop web service for protein bioinformatic analysis. It currently offers 34 interconnected external and in-house tools, whose functionality covers sequence similarity searching, alignment construction, detection of sequence features, structure prediction, and sequence classification. This breadth has made the Toolkit an important resource for experimental biology and for teaching bioinformatic inquiry. Recently, we replaced the first version of the Toolkit, which was released in 2005 and had served around 2.5 million queries, with an entirely new version, focusing on improved features for the comprehensive analysis of proteins, as well as on promoting teaching. For instance, our popular remote homology detection server, HHpred, now allows pairwise comparison of two sequences or alignments and offers additional profile HMMs for several model organisms and domain databases. Here, we introduce the new version of our Toolkit and its application to the analysis of proteins.

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem – limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis.

Differential expression of myometrial AP-1 proteins during gestation and labour.

Preterm labour (PTL) is a leading cause of perinatal mortality and postnatal morbidity. Contractions of the uterine muscle (myometrium) that determine the onset of labour depend on the expression of contraction‐associated proteins (CAPs, i.e. connexin43) regulated by dimeric AP‐1 transcription factors. Here, we examined subcellular (by immunoblotting) and tissue expression (by immunohistochemistry) of myometrial AP‐1 proteins (cJUN, JUNB, JUND, cFOS, FOSB, FRA1, FRA2) throughout gestation and TL in different species (mouse, rat and human). To identify the critical AP‐1 members associated with preterm birth, we studied their expression in mouse model of ‘infectious’ (LPS‐induced) and ‘sterile’ (RU486‐induced) PTL. We found that (1) myometrial AP‐1 composition is preserved in vivo between different species (rodents and human) indicating that Fos/Jun heterodimer (i.e. FRA2/JUND) may be indispensable for labour initiation. (2) Our in vivo study using murine models of gestation shows that there is a similarity in the myometrial AP‐1 protein composition during TL and pathological PTL of different aetiology suggesting the involvement of similar molecular machinery in the induction of labour. (3) This study is first comprehensive protein analysis of seven AP‐1 members in human labouring versus non‐labouring myometrium, showing their cellular expression and tissue distribution in relation to labour status.

Protein signatures of molecular pathways in non-small cell lung carcinoma (NSCLC): comparison of glycoproteomics and global proteomics

BackgroundNon-small cell lung carcinoma (NSCLC) remains the leading cause of cancer deaths in the United States. More than half of NSCLC patients have clinical presentations with locally advanced or metastatic disease at the time of diagnosis. The large-scale genomic analysis of NSCLC has demonstrated that molecular alterations are substantially different between adenocarcinoma (ADC) and squamous cell carcinoma (SqCC). However, a comprehensive analysis of proteins and glycoproteins in different subtypes of NSCLC using advanced proteomic approaches has not yet been conducted.MethodsWe applied mass spectrometry (MS) technology featuring proteomics and glycoproteomics to analyze six primary lung SqCCs and eleven ADCs, and we compared the expression level of proteins and glycoproteins in tumors using quantitative proteomics. Glycoproteins were analyzed by enrichment using a chemoenzymatic method, solid-phase extraction of glycopeptides, and quantified by iTRAQ-LC–MS/MS. Protein quantitation was further annotated via Ingenuity Pathway Analysis.ResultsOver 6000 global proteins and 480 glycoproteins were quantitatively identified in both SqCC and ADC. ADC proteins (8337) consisted of enzymes (22.11%), kinases (5.11%), transcription factors (6.85%), transporters (6.79%), and peptidases (3.30%). SqCC proteins (6967) had a very similar distribution. The identified glycoproteins, in order of relative abundance, included membrane (42%) and extracellular matrix (>33%) glycoproteins. Oncogene-coded proteins (82) increased 1.5-fold among 1047 oncogenes identified in ADC, while 124 proteins from SqCC were up-regulated in tumor tissues among a total of 827 proteins. We identified 680 and 563 tumor suppressor genes from ADC and SqCC, respectively.ConclusionOur systematic analysis of proteins and glycoproteins demonstrates changes of protein and glycoprotein relative abundance in SqCC (TP53, U2AF1, and RXR) and in ADC (SMARCA4, NOTCH1, PTEN, and MST1). Among them, eleven glycoproteins were upregulated in both ADC and SqCC. Two glycoproteins (ELANE and IGFBP3) were only increased in SqCC, and six glycoproteins (ACAN, LAMC2, THBS1, LTBP1, PSAP and COL1A2) were increased in ADC. Ingenuity Pathway Analysis (IPA) showed that several crucial pathways were activated in SqCC and ADC tumor tissues.

Proteogenomic analysis of the total and surface-exposed proteomes of Plasmodium vivax salivary gland sporozoites.

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum.

Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics.

Technological advances in liquid chromatography and tandem mass spectrometry (LC-MS/MS) have enabled comprehensive analyses of proteins and their post-translational modifications from cell culture and tissue samples. However, sample complexity necessitates offline prefractionation via a chromatographic method that is orthogonal to online reversed-phase high-performance liquid chromatography (RP-HPLC). This additional fractionation step improves target identification rates by reducing the complexity of the sample as it is introduced to the instrument. A commonly employed offline prefractionation method is high pH reversed-phase (Hi-pH RP) chromatography. Though highly orthogonal to online RP-HPLC, Hi-pH RP relies on buffers that interfere with electrospray ionization. Thus, samples that are prefractionated using Hi-pH RP are typically desalted prior to LC-MS/MS. In the present work, we evaluate an alternative offline prefractionation method, pentafluorophenyl (PFP)-based reversed-phase chromatography. Importantly, PFP prefractionation results in samples that are dried prior to analysis by LC-MS/MS. This reduction in sample handling relative to Hi-pH RP results in time savings and could facilitate higher target identification rates. Here, we have compared the performances of PFP and Hi-pH RP in offline prefractionation of peptides and phosphopeptides that have been isolated from human cervical carcinoma (HeLa) cells. Given the prevalence of isobaric mass tags for peptide quantification, we evaluated PFP chromatography of peptides labeled with tandem mass tags. Our results suggest that PFP is a viable alternative to Hi-pH RP for both peptide and phosphopeptide offline prefractionation.

Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry.

Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two-dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high-resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.