Characterization of effective, simple, and low-cost precipitation methods for depleting abundant plasma proteins to enhance the depth and breadth of plasma proteomics.

Plasma is an abundant source of proteins and potential biomarkers to aid in the detection, diagnosis, and prognosis of human diseases. These proteins are often present at low levels in the blood and difficult to identify and measure due to the large dynamic range of proteins. The goal of this work was to characterize and compare various protein precipitation methods related to how they affect the depth and breadth of plasma proteomic studies. Abundant protein precipitation with perchloric acid (PerCA) can increase protein identifications and depth of plasma proteomic studies. Three acid- and four solvent-based precipitation methods were evaluated. All methods tested provided excellent plasma proteomic coverage (>600 identified protein groups) and detected protein in the low pg/mL range. Functional enrichment analysis revealed subtle differences within and larger changes between the precipitant groups. Methanol-based precipitation outperformed the other methods based on identifications and reproducibility. The methods' performance was verified using eight lung cancer patient samples, where >700 protein groups were measured and proteins with an estimated plasma concentration of ∼10 pg/mL were detected. Various protein precipitation agents are amenable to extending the depth and breadth of plasma proteomes. These data can guide investigators to implement inexpensive, high-throughput methods for their plasma proteomic workflows.

Multiplex Protein Biomarker Profiling in Patients with Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH), is an autosomal dominant disorder caused by mutations in the LDLR, APOB, PCSK9, and APOE genes and is characterized by high plasma levels of total and low-density lipoprotein (LDL) cholesterol. Our study aimed to analyze the influences of two different therapies on a wide spectrum of plasma protein biomarkers of cardiovascular diseases. Plasma from FH patients under hypolipidemic therapy (N = 18; men = 8, age 55.4 ± 13.1 years) and patients under combined long-term LDL apheresis/hypolipidemic therapy (N = 14; men = 7; age 58.0 ± 13.6 years) were analyzed in our study. We measured a profile of 184 cardiovascular disease (CVD) associated proteins using a proximity extension assay (PEA). Hypolipidemic therapy significantly (all p < 0.01) influenced 10 plasma proteins (TM, DKK1, CCL3, CD4, PDGF subunit B, AGRP, IL18, THPO, and LOX1 decreased; ST2 increased). Under combined apheresis/hypolipidemic treatment, 18 plasma proteins (LDLR, PCSK9, MMP-3, GDF2, CTRC, SORT1, VEGFD, IL27, CCL24, and KIM1 decreased; OPN, COL1A1, KLK6, IL4RA, PLC, TNFR1, GLO1, and PTX3 increased) were significantly affected (all p < 0.006). Hypolipidemic treatment mainly affected biomarkers involved in vascular endothelial maintenance. Combined therapy influenced proteins that participate in cholesterol metabolism and inflammation.

Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma.

Cancer cells secrete membranous extracellular vesicles (EVs) which contain specific oncogenic molecular cargo (including oncoproteins, oncopeptides, and RNA) into their microenvironment and the circulation. As such, EVs including exosomes (small EVs) and microvesicles (large EVs) represent important circulating biomarkers for various diseases, including cancer and its progression. These circulating biomarkers offer a potentially minimally invasive and repeatable targets for analysis (liquid biopsy) that could aid in the diagnosis, risk stratification, and monitoring of cancer. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remain challenging. Like EVs, other types of circulating biomarkers (including cell-free nucleic acids, cf-NAs; or circulating tumor cells, CTCs) may represent a complementary or alternative approach to cancer diagnosis. In the context of multiple myeloma (MM), a systemic cancer type that causes cancer cells to accumulate in the bone marrow, the specific role for EVs as biomarkers for diagnosis and monitoring remains undefined. Tumor heterogeneity along with the various subtypes of MM (such as non-secretory MM) that cannot be monitored using conventional testing (e.g. sequential serological testing and bone marrow biopsies) render liquid biopsy and circulating tumor-derived EVs a promising approach. In this protocol, we describe the isolation and purification of EVs from peripheral blood plasma (PBPL) collected from healthy donors and patients with MM for a biomarker discovery strategy. Our results demonstrate detection of circulating EVs from as little as 1 mL of MM patients' PBPL. High-resolution mass spectrometry (MS)-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease. We describe biophysical characterization and quantitative proteomic profiling of disease-specific circulating EVs which may provide important implications for the development of cancer diagnostics in MM.

Identification of biomarkers for drug-resistant endometriosis using clinical proteomics.

Dienogest (DNG), is an effective and widely used progestin used in the treatment of endometriosis, yet clinically, a subset of cases show resistance to DNG treatment. During a previous investigation on the effect of DNG of cytokines and growth factor production, we incidentally found that endometriotic cyst fluid did not demonstrate inhibitory effects to DNG in a subset of cases. To clarify the mechanisms of this resistance to DNG, we performed proteomics analysis to compare the protein expression between DNG-sensitive and resistant cases. Based upon our results, several proteins were extracted that relate to neutrophil granulocyte activation marker (myeloperoxidase, lactotransferrin), inflammation (azurocidin, neutrophil gelatinase-associated lipocalin, etc.), and others biological processes reflecting the clinical environment of the endometriotic cyst. Among these proteins, azurocidin (AZU) is perhaps most interesting one as azurocidin is a protease that cleaves insulin-like growth factor-1 (IGFBP-1) associated with clear cell carcinoma of the ovary. We propose that the proteins extracted in the present study warrant further investigation in their relationship to carcinogenesis of endometrioma.

Galectin-3 is modulated in pancreatic cancer cells under hypoxia and nutrient deprivation.

Pancreatic ductal adenocarcinoma is one of the most aggressive tumors with a microenvironment marked by hypoxia and starvation. Galectin-3 has been evaluated in solid tumors and seems to present both pro/anti-tumor effects. So, this study aims to characterize the expression of Galectin-3 from pancreatic tumor cells and analyze its influence for cell survive and motility in mimetic microenvironment. For this, cell cycle and cell death were accessed through flow cytometry. Characterization of inside and outside Galectin-3 was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence, Western blot, and ELISA. Consequences of Galectin-3 extracellular inhibition were investigated using cell death and scratch assays. PANC-1 showed increased Galectin-3 mRNA expression when cultivated in hypoxia for 24 and 48 h. After 24 h in simultaneously hypoxic/deprived incubation, PANC-1 shows increased Galectin-3 protein and secreted levels. For Mia PaCa-2, cultivation in deprivation was determinant for the increasing in Galectin-3 mRNA expression. When cultivated in simultaneously hypoxic/deprived condition, Mia PaCa-2 also presented increasing for the Galectin-3 secreted levels. Treatment of PANC-1 cells with lactose increased the death rate when cells were incubated simultaneously hypoxic/deprived condition. Therefore, it is possible to conclude that the microenvironmental conditions modulate the Galectin-3 expression on the transcriptional and translational levels for pancreatic cancer cells.

Pyrolyzed cotton balls for protein removal: Analysis of pharmaceuticals in serum by capillary electrophoresis.

Because of the inherent affinity of proteins for bare, fused silica capillaries, the analysis of protein-containing samples has proven a challenging task for capillary electrophoresis. The adsorption of proteins to the capillary walls effectively changes the zeta potential and thus affects the electro-osmotic flow leading to significant shifts in migration time, peak broadening, and poor reproducibility. While there are several well-known methods to remove proteins from samples prior to the analysis (including precipitation) or to prevent their adsorption to the capillary (semi-permanent coatings), those approaches are often expensive, time consuming, or simply unreliable. Aiming to address these needs, this manuscript reports on the use of pyrolyzed cotton balls, as a simple and widely accessible hydrophobic material to remove proteins from serum samples. The material retains enough flexibility so it can be placed directly into the sample vials and has enough capacity to capture more than 75% of the proteins in the sample (1% dilution of 1 mL of serum). The advantages of the material are demonstrated by performing the analysis of five representative drugs (in serum) by capillary electrophoresis obtaining a change in migration time of only 5 ± 1%, after 10 consecutive runs.

Purification and Assays of Tachylectin-5.

Tachylectin-5, a 41-kDa protein with a common fold of the C-terminal globular domain of the γ-chain of fibrinogen, is purified from horseshoe crab hemolymph plasma by affinity column chromatography, using acetyl-group-immobilized resin. Two types of isolectins, tachylectin-5A and tachylectin-5B, are obtained by stepwise elution with GlcNAc at 25 and 250 mM, respectively. Tachylectins-5A and -5B exhibit extraordinarily strong hemagglutinating activity against all types of human erythrocytes (the minimum agglutinating concentration of 0.004-0.008 µg/mL for tachylectin-5A and 0.077-0.27 µg/mL for tachylectin-5B). Their hemagglutinating activities are inhibited by acetyl group-containing sugars and noncarbohydrates such as sodium acetate, acetylcholine, and acetyl CoA (the minimum inhibitory concentrations of 1.3-1.6 mM), indicating that the acetyl group is required and sufficient for recognition by tachylectins-5A and -5B. EDTA inhibits their hemagglutinating activity, whereas the inhibition is overcome by adding an excess amount of Ca2+. Tachylectins-5A and -5B also exhibit bacterial agglutinating activity against both Gram-negative bacteria (the minimum agglutinating concentrations of 0.04-0.08 µg/mL for tachylectin-5A and 0.05-0.11 µg/mL for tachylectin-5B) and Gram-positive bacteria (the minimum agglutinating concentrations of 0.3-2.4 µg/mL for tachylectin-5A and 15.1-26.8 µg/mL for tachylectin-5B). Interestingly, tachylectins-5A and -5B enhance the antimicrobial activity of a hemocyte-derived peptide, big defensin.

Purification and Assays of Tachycitin.

An antimicrobial peptide tachycitin (73 amino acids) is purified by steps of chromatography, including Sephadex G-50 and S Sepharose FF, from the acid extract of hemocyte debris of horseshoe crabs. Tachycitin is present in monomer form in solution, revealed by ultracentrifugation analysis. Tachycitin exhibits bacterial agglutination activity and inhibits the growth of both Gram-negative bacteria, Gram-positive bacteria, and fungus Candida albicans. Interestingly, tachycitin shows synergistic antimicrobial activity in corporation with another antimicrobial peptide, big defensin. Tachycitin shows a specific binding activity to chitin but not to cellulose, mannan, xylan, and laminarin. Tachycitin is composed of the N-terminal three-stranded ß-sheet and the C-terminal two-stranded ß-sheet following a short helical turn, and the C-terminal structural motif shares a significant structural similarity with the chitin-binding domain derived from a plant chitin-binding protein, hevein.

SEC hyphenated to a multielement-specific detector unravels the degradation pathway of a bimetallic anticancer complex in human plasma.

The bimetallic metal complex Titanocref exhibits relevant anticancer activity, but it is unknown if it is stable to reach target tissues intact. To gain insight, a pharmacologically relevant dose was added to human blood plasma and the mixture was incubated at 37 °C. The obtained mixture was analyzed 5 and 60 min later by size-exclusion chromatography hyphenated to an inductively coupled plasma atomic emission spectrometer (SEC-ICP-AES). We simultaneously detected several titanium (Ti), gold (Au) and sulfur (S)-peaks, which corresponded to a Ti degradation product that eluted partially, and a Au degradation product that eluted entirely bound to plasma proteins (both time points). Although ~70% of the intact Titanocref was retained on the column after 60 min, our results allowed us to establish - for the first time - its likely degradation pathway in human plasma at near physiological conditions. These results suggest that ~70% of Titanocref remain in plasma after 60 min, which supports results from a recent in vivo study in which mice were treated with Titanocref and revealed Ti:Au molar ratios in tumors and organs close to 1:1. Thus, our stability studies suggest that the intact drug is able to reach target tissue. Overall, our results exemplify that SEC-ICP-AES enables the execution of intermediate in vitro studies with human plasma in the context of advancing bimetallic metal-based drugs to more costly clinical studies.

Purification and identification of antioxidant peptides from duck plasma proteins.

The antioxidant peptides extracted from duck plasma hydrolysate (DPH) was investigated. The antioxidant activity of DPH, which was isolated and purified via ultrafiltration, size exclusion chromatography, and reversed-phase high-performance liquid chromatography, was evaluated using its free radical scavenging ability. Nano-liquid chromatography-tandem mass spectrometry was conducted to identify the DPH fractions with the highest antioxidant ability. Seven novel peptides: LDGP, TGVGTK, EVGK, RCLQ, LHDVK, KLGA, and AGGVPAG (400.43, 561.63, 431.48, 260.14, 610.71, 387.47, and 527.57 Da, respectively) were identified and synthesized using a solid-phase peptide produce to evaluate their antioxidant activities. Of these, EVGK exhibited the highest Fe2+ chelating ability (16.35%), and RCLQ presented the highest reducing power, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt scavenging activity, and 1,1-diphenyl-2-picrylhydrazyl scavenging rate (0.62, 274.83 mM TE/mg, and 95.12%, respectively). Our results indicated that DPH possessed antioxidant capabilities and could be used to obtain antioxidant peptides, thus adding economic value to duck blood.

Interfacial charge regulation of protein blocking layers in transistor biosensor for direct measurement in serum.

Ion-sensitive field-effect transistor (ISFET) as a biosensor facilitates a process of data-acquisition through label-free and real-time monitoring. Direct quantification of a biomarker in serum is challenging in ISFET biosensor since charged proteins in serum interfere transduction to electrical signals. Here, we report the fabrication of protein blocking layers (PBLs) with intended interfacial charges to minimize non-specific protein bindings on ISFET. Use of charged protein precursors enables to regulate the interfacial charge of PBLs, preserving their intrinsic electric features (neutral: hemoglobin, positively charged: lysozyme, negatively charged: BSA). The effect of this interfacial charge on the signal was demonstrated through PSMA (prostate cancer biomarker) sensing using a dual-gate ISFET biosensor. The neutral PBL showed the minimum noise compared to the negatively and positively charged PBLs, enabling the ISFET to exhibit the same detection range in untreated serum as with pre- or post-treatment (1 fg/ml to 100 ng/ml). The introduction of neutral PBLs to ISFET biosensors would allow the application of the ISFET biosensor as a point-of-care device.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles.

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa-casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein-protein interactions, which can affect its activity.

Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics.

Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

The Ameliorating Effect of Plasma Protein from Tachypleus tridentatus on Cyclophosphamide-Induced Acute Kidney Injury in Mice.

In the study, the protective effect of plasma protein from Tachypleus tridentatus (PPTT) on acute kidney injury (AKI) and the related molecular mechanisms were first investigated by Western blotting analyses, TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry. It was found that PPTT had an obviously inhibitory effect on Reactive oxygen species (ROS) in cyclophosphamide (CTX)-exposed mice. Furthermore, results demonstrated that the renal cell death mode is due to inducing apoptosis and autophagy inhibited by dose-dependent PPTT in mice treated with CTX by decreasing the protein expression of bax, beclin-1, and LC3 and increasing the expression of bcl-2. Moreover, the p38 MAPK and PI3K/Akt signaling pathways were observed to take part in the PPTT-induced renal cell growth effect by enhancing the upregulation of the expression of Akt and p-Akt as well as the downregulation of the expression of p38 and p-p38, which indicated a PPTT ameliorating effect on AKI CTX-induced in mice through p38 MAPK and PI3K/Akt signaling pathways. Briefly, this article preliminarily studies the mechanism of the PPTT ameliorating effect on AKI CTX-induced in mice, which helps to provide a reference for PPTT clinical application in AKI therapy.

Regenerative NanoOctopus Based on Multivalent-Aptamer-Functionalized Magnetic Microparticles for Effective Cell Capture in Whole Blood.

Isolation of specific rare cell subtypes from whole blood is critical in cellular analysis and important in basic and clinical research. Traditional immunomagnetic cell capture suffers from suboptimal sensitivity, specificity, and time- and cost-effectiveness. Mimicking the features of octopuses, a device termed a "NanoOctopus" was developed for cancer cell isolation in whole blood. The device consists of long multimerized aptamer DNA strands, or tentacle DNA, immobilized on magnetic microparticle surfaces. Their ultrahigh sensitivity and specificity are attributed to multivalent binding of the tentacle DNA to cell receptors without steric hindrance. The simple, quick, and noninvasive capture and release of the target cells allows for extensive downstream cellular and molecular analysis, and the time- and cost-effectiveness of fabrication and regeneration of the devices makes them attractive for industrial manufacture.

Mouse serum protects against total body irradiation-induced hematopoietic system injury by improving the systemic environment after radiation.

Reactive oxygen species (ROS) play a critical role in total body irradiation (TBI)-induced hematopoietic system injury. However, the mechanisms involved in ROS production in hematopoietic stem cells (HSCs) post TBI need to be further explored. In this study, we demonstrated that hematopoietic system injury in mice radiated with TBI was effectively alleviated when the blood circulation environment was changed via the injection of serum from non-radiated mice. Serum injection increased the survival of radiated mice and ameliorated TBI-induced hematopoietic system injury through attenuating myeloid skew, increasing HSC frequency, and promoting the reconstitution of radiated HSCs. Serum injection also decreased ROS levels in HSCs and regulated oxidative stress-related proteins. A serum proteome sequence array showed that proteins related to tissue injury and oxidative stress were regulated, and a serum-derived exosome microRNA sequence assay showed that the PI3K-Akt and Hippo signaling pathways were affected in radiated mice injected with serum from non-radiated mice. Furthermore, a significant increase in cell viability and a decrease in ROS were observed in radiated lineage-c-kit+ cells treated with serum-derived exosomes. Similarly, an improvement in the impaired differentiation of HSCs was observed in radiated mice injected with serum-derived exosomes. Taken together, our observations suggest that serum from non-radiated mice alleviates HSC injury in radiated mice by improving the systemic environment after radiation, and exosomes contribute to this radioprotective effect as important serum active component.

Comparison of an Optimized Ultracentrifugation Method versus Size-Exclusion Chromatography for Isolation of Exosomes from Human Serum.

Exosomes are nanosized vesicles that are abundant in biological fluids. In recent years, exosomes have attracted increasing attention as their cargo may provide promising biomarkers for the early diagnosis of and therapy for many diseases, such as cancer. In addition to ultracentrifugation (UC), many alternative methods including size-exclusion chromatography (SEC) have been developed for isolating exosomes. It has been reported that the SEC method provided improved performance relative to the UC method in isolating exosomes from plasma, where the former contained less residual blood protein contamination. We have compared the SEC method with an optimized UC method in isolating exosomes from human serum. This was based on dilution of the serum to reduce the viscosity and a prolonged cycle of UC, followed by another four cycles. We found that >95% of serum proteins were removed without a significant loss of exosome proteins relative to SEC. We also combined one cycle of UC with SEC and found that this method provided improved results relative to the SEC method, although the serum protein contamination was several times higher than that of our optimized UC method. The TEM showed that the size distribution of exosomes isolated from each of the three methods was similar.

Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.