Immunoaffinity depletion with antibodies to the top 7 or 14 high-abundance plasma proteins is used enhance detection of lower abundance in both shotgun and targeted proteomic analyses. We evaluated effects 7/top immunodepletion on analysis human plasma. Our goal was evaluate impact across detectable ranges abundance. The columns afforded highly repeatable efficient protein fractionation. Relatively few nontargeted were captured by columns. Analyses unfractionated immunodepleted peptide...

Reproducible quantification of large biological cohorts is critical for clinical/pharmaceutical proteomics yet remains challenging because most prevalent methods suffer from drastically declined commonly quantified proteins and substantially deteriorated quantitative quality as cohort size expands. MS2-based data-independent acquisition approaches represent tremendous advancements in reproducible protein measurement, but often with limited depth. We developed IonStar, an MS1-based approach...

In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar strategy achieving excellent quantitative quality of MS1 quantification. Key features...

Proteomic analysis of bronchoalveolar lavage fluid (BALF) in chronic obstructive pulmonary disease (COPD) patients may provide new biomarkers and deeper understanding the mechanisms but remains challenging. Here we describe an ion-current-based strategy for comparative BALF proteomes from with moderate stable COPD versus healthy controls. The includes efficient preparation procedure providing quantitative recovery a nano-LC/MS long, heated column. Under optimized conditions, high efficiency...

Age-related macular degeneration (AMD) is the leading cause of blindness among elderly. Currently, there are no treatments for dry AMD, which characterized by death retinal pigment epithelium (RPE) and photoreceptors. Reports from human donors with AMD suggest that RPE mitochondrial defects a key event in pathology. Thus, most effective strategy treating to identify compounds enhance function subsequently, preserve RPE. In this study, primary cultures (n = 20) or without 8) were used...

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and characterized by multiple congenital malformations as well neurosensory cognitive abnormalities. A rat model SLOS has been developed that exhibits progressive retinal degeneration visual dysfunction; however, molecular events underlying dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from age- sex-matched control rats (n =...

For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as often required by clinical/pharmaceutical studies. We describe a rapid straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior organic solvent precipitation on-pellet digestion, SEPOD treats samples cocktail (SC) containing multiple...

Survey-scan-based label-free method have shown no compelling benefit over fragment ion (MS2)-based approaches when low-resolution mass spectrometry (MS) was used, the growing prevalence of high-resolution analyzers may changed game. This necessitates an updated, comparative investigation these for data acquired by MS. Here, we compared survey scan-based (ion current, IC) and MS2-based abundance features including spectral-count (SpC) MS2 total-ion-current (MS2-TIC), quantitative analysis...

Osteoclasts are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In vitro models principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 frequently used because they widely available, easy culture, more amenable genetic manipulation than cells. Increasing evidence, however, has shown that the vastly different origins these two cell types may...

Regulators of G-protein Signaling are a conserved family proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion vivo protected mice against pathological bone loss. To characterize mechanism osteoclastogenesis, we selectively deleted C57BL/6J targeting precursors using LyzM-driven Cre or overexpressed RAW264.7 cells. led to an osteopetrotic phenotype evidenced by...

The plasma proteome holds enormous clinical potential, yet an in-depth analysis of the remains a daunting challenge due to its high complexity and extremely wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable animal proteome, which is often essential experimental pathology/pharmacology. Here we describe highly comprehensive method investigation employs optimized combinatorial peptide ligand...

A proteome-level time-series study of drug effects (i.e., pharmacodynamics) is critical for understanding mechanisms action and systems pharmacology, but challenging, because the requirement a proteomics method reliable quantification many biological samples. Here, we describe highly reproducible strategy, enabling global, large-scale investigation expression dynamics corticosteroid-regulated proteins in livers from adrenalectomized rats over 11 time points after dosing (0.5-66 h, N =...

The two key steps for analyzing proteomic data generated by high-resolution MS are database searching and postprocessing. While the interrelated, studies on their combinatory effects optimization of these procedures have not been adequately conducted. Here, we investigated performance three popular search engines (SEQUEST, Mascot, Amanda) in conjunction with five filtering approaches, including respective score-based filtering, a group-based approach, local false discovery rate (LFDR),...

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS CS-FBS may reduce the activity of receptor (AR), inhibit cell proliferation, or modulate intracellular metabolism. The removal proteins by charcoal stripping cause changes biological functions has not yet been investigated. Here we profiled using an ion-current-based quantitative platform consisting reproducible...

Proteomics analysis of circulating exosomes derived from cancer cells represents a promising approach to the elucidation cell–cell communication and discovery putative biomarker candidates for diagnosis treatment. Nonetheless, proteome cell lines with different metastatic capabilities still warrants further investigation. Here, we present comprehensive quantitative proteomics investigation isolated immortalized mammary epithelial matched tumor potentials in an attempt discover exosome...

Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis retinopathy prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore proteomic changes in early stage ROP rat model oxygen-induced (OIR). Retina proteins, which are rich membrane were efficiently extracted by detergent-cocktail and subjected precipitation/on-pellet-digestion, followed nano-LC-MS analysis on...

Trypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community explore various unique aspects of T. biology in search better treatments. In this study, we identified protein arginine methyltransferase (PRMT), Tb PRMT1, factor that modulates numerous biology. These include glycolysis life cycle...

The recently-introduced Orbitrap Fusion mass spectrometry permits various types of MS2 acquisition methods. To date, these different strategies and the optimal data interpretation approach for each have not been adequately evaluated. This study comprehensively investigated four strategies: HCD-OT (higher-energy-collisional-dissociation with detection), HCD-IT (HCD ion trap, IT), CID-IT (collision-induced-dissociation IT) CID-OT on Fusion. achieve extensive comparison identify method...

Immobilized pH gradients isoelectric focusing (IPG-IEF) is the first dimension typically used in two-dimensional gel electrophoresis (2-DE). It can also be on its own conjunction with tandem mass spectrometry (MS/MS) for analysis of proteins. Here, we described a strategy combining immobilized gradient strips, and to create new high-throughput sensitive detection method. Protein mixture separated by in-gel IEF, then entire strip cut into set sections. Proteins each section are digested...

Dietary isothiocyanates (ITCs) from cruciferous vegetables have shown potent anti-breast cancer activities in preclinical models, but their anticancer effects vivo breast patients remain elusive. A proof-of-principle, presurgical window of opportunity trial is conducted to assess the dietary ITCs patients.

The rapidly expanding availability of high-resolution mass spectrometry has substantially enhanced the ion-current-based relative quantification techniques. Despite increasing interest in methods, quantitative sensitivity, accuracy, and false discovery rate remain major concerns; consequently, comprehensive evaluation development these regards are urgently needed. Here we describe an integrated, new procedure for data normalization protein ratio estimation, termed ICan, improved analysis...

Corticosteroids (CS) such as methylprednisolone (MPL) affect almost all liver functions through multiple mechanisms of action, and long-term use results in dysregulation causing diverse side effects. The complexity involved molecular necessitates a systems approach. Integration information from the transcriptomic proteomic responses has potential to provide deeper insights into CS actions. present report describes tandem analysis rich time-series data rat after single dose MPL. Hierarchical...