In Vivo Mechanism of Action of Sodium Caprate for Improving the Intestinal Absorption of a GLP1/GIP Coagonist Peptide.

Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY's favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations.

Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.

Proximal Tubules Have the Capacity to Regulate Uptake of Albumin.

Evidence from multiple studies supports the concept that both glomerular filtration and proximal tubule (PT) reclamation affect urinary albumin excretion rate. To better understand these roles of glomerular filtration and PT uptake, we investigated these processes in two distinct animal models. In a rat model of acute exogenous albumin overload, we quantified glomerular sieving coefficients (GSC) and PT uptake of Texas Red-labeled rat serum albumin using two-photon intravital microscopy. No change in GSC was observed, but a significant decrease in PT albumin uptake was quantified. In a second model, loss of endogenous albumin was induced in rats by podocyte-specific transgenic expression of diphtheria toxin receptor. In these albumin-deficient rats, exposure to diphtheria toxin induced an increase in albumin GSC and albumin filtration, resulting in increased exposure of the PTs to endogenous albumin. In this case, PT albumin reabsorption was markedly increased. Analysis of known albumin receptors and assessment of cortical protein expression in the albumin overload model, conducted to identify potential proteins and pathways affected by acute protein overload, revealed changes in the expression levels of calreticulin, disabled homolog 2, NRF2, angiopoietin-2, and proteins involved in ATP synthesis. Taken together, these results suggest that a regulated PT cell albumin uptake system can respond rapidly to different physiologic conditions to minimize alterations in serum albumin level.

Pyruvate dehydrogenase alpha 1 as a target of omega-3 polyunsaturated fatty acids in human prostate cancer through a global phosphoproteomic analysis.

Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein-enrichment strategy and high-resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO- or oleic acid-treated groups uncovered a number of important protein phosphorylation changes induced by n-3PUFAs. This phosphoproteomic discovery study and the follow-up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine-232 and serine-300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO-induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n-3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation.

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice.

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.

Identification of novel biomarker candidates for immunohistochemical diagnosis to distinguish low-grade chondrosarcoma from enchondroma.

Chondrosarcoma is the third most common primary bone cancer, requiring surgical resection. However, differentiation of low-grade chondrosarcoma (grade 1) from enchondroma that is benign and only requires regular follow-up is one of the most frequent diagnostic dilemmas facing orthopedic oncologists in clinical management. Although multiple techniques are applied to make the distinction, immunohistochemistry is an important ancillary technique, especially when a histopathological stain of specimen must be obtained in order to guarantee an accurate confirmation. Currently, no adequate immunohistochemical diagnostic protein biomarkers are available to distinguish low-grade chondrosarcoma from enchondroma. To discover novel protein biomarker candidates, an LC-MS/MS approach was applied to directly compare formalin-fixed, paraffin-embedded low-grade chondrosarcoma with enchondroma tissue samples. The proteomics analysis revealed 17 protein biomarker candidates. A principle was developed to prioritize the candidates using category and ranking. An algorithm, prioritization index of biomarker candidates for immunohistochemistry on tissue specimens, was developed to rank the candidates inside each category. Using the proteomics data and bioinformatics results, the prioritization index of biomarker candidates for immunohistochemistry on tissue revealed periostin as a top candidate. Immunohistochemical staining of periostin in 23 low-grade chondrosarcoma and 31 enchondroma tissue specimens disclosed 87% specificity and 70% sensitivity.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery.

PURPOSE: To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS: In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS: Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS: Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways.

Proteomic profiling of human sera for discovery of potential biomarkers to monitor abstinence from alcohol abuse.

Although numerous biomarkers or biomarker candidates have been discovered to detect levels of drinking and intervals of time after last drinking episode, only a few biomarkers have been applied to monitor abstinence in a longer interval (≥6 wks) from alcohol abuse. Considering sample sources, sensitivity, and specificity, new biomarkers from blood with better accuracy are needed. To address this, serum proteomic profiles were compared between pre- and post- treatment samples from subjects seeking treatment for alcohol abuse and dependence in an intensive 6 wk daily outpatient program using high-abundance plasma protein immunodepletion and LC-MS/MS techniques. Protein identification, quantification, candidate biomarker selection, and prioritization analyses were carried out. Among the 246 quantified serum proteins, abundance of 13 and 45 proteins in female and male subjects were significantly changed (p ≤ 0.05), respectively. Of these biomarker candidate proteins, 2 (female) and 8 (male) proteins were listed in category 1, with high area under the receiver operating characteristic curve, sensitivity, specificity, and fold change. In summary, several new biomarker candidates have been identified to monitor abstinence from alcohol abuse.

Novel serum biomarkers for detection of excessive alcohol use.

BACKGROUND: Construct interview that correctly identifies those with alcohol use disorder have limitation, especially when the subjects are motivated to minimize the magnitude of drinking behavior. Current laboratory tests to detect excessive alcohol consumption are limited by marginal sensitivity/specificity. Excessive drinking has been shown to affect several organ systems, which may be reflected in changes in quantity of plasma proteins. Our aim was to employ novel proteomic analyses to identify potential markers for excessive alcohol use. METHODS: A prospective case-control study included 49 controls and 54 excessive drinkers (discovery cohort). The serum proteomic analyses in these subjects were performed, and the results were tested in the verification cohort (40 controls and 40 excessive drinkers). RESULTS: Using the appropriate cutoff and confirmation with ELISA, we identified 4 proteins which were significantly elevated in the serum of excessive drinkers: AT-rich interactive domain-containing protein 4B (ARID4B), phosphatidylcholine-sterol acyltransferase (LCAT), hepatocyte growth factor-like protein (MST1), and ADP-ribosylation factor 6 (ARL6). The performance of the conventional markers (aspartate aminotransferase [AST], alanine aminotransferase [ALT], gamma-glutamyl transpeptidase [GGT], percentage of carbohydrate-deficient transferrin [%CDT], and mean corpuscular volume [MCV]) discriminating between excessive alcohol use and controls had an area under the curve (AUC) ranging from 0.21 (ALT) to 0.67 (MCV). The AUC of these novel proteins showed the improvement in the detection of excessive drinkers compared to conventional laboratory tests, ranging from 0.73 (for ARID4B) to 0.86 (for ARL6). CONCLUSIONS: We have identified 4 novel proteins that can discern subjects with excessive alcohol use. Further studies are needed to determine the clinical implications of these markers to detect excessive alcohol use and confirm abstinence.

Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis.

Because fresh-frozen tissue samples associated with long-term clinical data and of rare diseases are often unobtainable at the present time, formalin-fixed paraffin-embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross-link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 µg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97-99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high-yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic.

Perivascular adipose tissue potentiates contraction of coronary vascular smooth muscle: influence of obesity.

BACKGROUND: This investigation examined the mechanisms by which coronary perivascular adipose tissue (PVAT)-derived factors influence vasomotor tone and the PVAT proteome in lean versus obese swine. METHODS AND RESULTS: Coronary arteries from Ossabaw swine were isolated for isometric tension studies. We found that coronary (P=0.03) and mesenteric (P=0.04) but not subcutaneous adipose tissue augmented coronary contractions to KCl (20 mmol/L). Inhibition of CaV1.2 channels with nifedipine (0.1 µmol/L) or diltiazem (10 µmol/L) abolished this effect. Coronary PVAT increased baseline tension and potentiated constriction of isolated arteries to prostaglandin F2α in proportion to the amount of PVAT present (0.1-1.0 g). These effects were elevated in tissues obtained from obese swine and were observed in intact and endothelium denuded arteries. Coronary PVAT also diminished H2O2-mediated vasodilation in lean and, to a lesser extent, in obese arteries. These effects were associated with alterations in the obese coronary PVAT proteome (detected 186 alterations) and elevated voltage-dependent increases in intracellular [Ca(2+)] in obese smooth muscle cells. Further studies revealed that the Rho-kinase inhibitor fasudil (1 µmol/L) significantly blunted artery contractions to KCl and PVAT in lean but not obese swine. Calpastatin (10 µmol/L) also augmented contractions to levels similar to that observed in the presence of PVAT. CONCLUSIONS: Vascular effects of PVAT vary according to anatomic location and are influenced by an obese phenotype. Augmented contractile effects of obese coronary PVAT are related to alterations in the PVAT proteome (eg, calpastatin), Rho-dependent signaling, and the functional contribution of K(+) and CaV1.2 channels to smooth muscle tone.

Using long columns to quantify over 9200 unique protein groups from brain tissue in a single injection on an Orbitrap Exploris 480 mass spectrometer.

The most exciting advancement in LC-MS/MS-based bottom-up proteomics has centered around enhancing mass spectrometers. Among these, the latest and most advanced mass spectrometer for bottom-up proteomics is the Orbitrap Astral that has the highest scan rate to accelerate throughput and the highest sensitivity to handle a very small amount of peptide samples and to achieve deeper proteomics. However, its affordability remains a challenge for most laboratories. While significant strides have been made in improving mass spectrometry, advancing liquid chromatography (LC) to achieve deeper proteomics has not achieved significant successes since the innovation of Multidimensional Protein Identification Technology (MudPIT) in 2001. To achieve deeper proteomics in a less labor-intensive and more reproducible approach while using a more cost-effective mass spectrometer, such as the Orbitrap Exploris 480, we evaluated trap columns as long as 40 cm and analytical column as long as 600 cm besides sample loading amount, gradient time, and analytical column particle size to enable a fractionation-free method for a single injection to obtain deeper proteomics. The length of trap and analytic columns is the key factor. Using a 30 cm trap column and 250 cm analytical column with other optimized LC conditions, we quantified over 9200 unique protein groups from brain tissue in a single injection using a 24-h gradient on an Orbitrap Exploris 480 mass spectrometer.

Pursuing Impactful Quantitative Proteomics Using QC-Channels in Every Spectrum and Trend-Design in Experiment.

False changes discovered by quantitative proteomics reduce the trust of biologists in proteomics and limit the applications of proteomics to unlock biological mechanisms, which suppresses the application of proteomics techniques in the pharmaceutical industry more than it does in academic research. To remove false changes that arise during LC-MS/MS data acquisition, we evaluated the contributions of peptide abundance and number of unique peptides on reproducibility. Lower abundance and only one unique peptide have a higher risk of generating a higher coefficient of variation (CV), resulting in less accurate quantification. However, the abundance of peptides in samples is not adjustable and discarding proteins quantified by only one unique peptide is not a choice either. Indeed, a large percentage of proteins are accurately quantified by only one unique peptide. Therefore, to improve the calculations of the CV, we leverage a new function in PEAKS called QC-channels which enables technical replicates of each spectrum to be evaluated prior to calculation of the CV. While the QC-channels function in PEAKS significantly reduced the false quantification, random false changes still exist due to known or unknown reasons. To address this challenge, we present the idea of Trend-design to track trend changes rather than changes from two points to remove false quantifications and reveal consequential changes responding to a treatment or condition. The idea was confirmed by molecules with different affinity and dose in the current study. The combination of QC-channels and Trend-design enables a more impactful quantitative proteomics to allow unlocking biological mechanisms using proteomics.

Proteomic analysis of hearts from Akita mice suggests that increases in soluble epoxide hydrolase and antioxidative programming are key changes in early stages of diabetic cardiomyopathy.

Cardiovascular disease is the leading cause of diabetic morbidity with more than 10% of type 1 diabetes mellitus (T1DM) patients dying before they are 40 years old. This study utilized Akita mice, a murine model with T1DM progression analogous to that of humans. Diabetic cardiomyopathy in Akita mice presents as cardiac atrophy and diastolic impairment at 3 months of age, but we observed cardiac atrophy in hearts from recently diabetic mice (5 weeks old). Hearts from 5 week old mice were analyzed with a rigorous label-free quantitative proteomic approach to identify proteins that may play a critical role in the early pathophysiology of diabetic cardiomyopathy. Eleven proteins were differentially expressed in diabetic hearts: products of GANC, PLEKHN1, COL1A1, GSTK1, ATP1A3, RAP1A, ACADS, EEF1A1, HRC, EPHX2, and PKP2 (gene names). These proteins are active in cellular defense, metabolism, insulin signaling, and calcium handling. Further analysis of Akita hearts using biochemical assays showed that the cellular defenses against oxidative stress were increased, including antioxidant capacity (2-3-fold) and glutathione levels (20%). Immunoblots of five and twelve week old Akita heart homogenates showed 30% and 145% increases in expression of soluble epoxide hydrolase (sEH (gene name EPHX2)), respectively, and an approximate 100% increase in sEH was seen in gastrocnemius tissue of 12 week old Akita mice. In contrast, 12 week old Akita livers showed no change in sEH expression. Our results suggest that increases in sEH and antioxidative programming are key factors in the development of type 1 diabetic cardiomyopathy in Akita mice and reveal several other proteins whose expression may be important in this complex pathophysiology.

Comparison of nanotube-protein corona composition in cell culture media.

In biological environments, nanomaterials associate with proteins forming a protein corona (PC). The PC may alter the nanomaterial's pharmacokinetics and pharmacodynamics, thereby influencing toxicity. Using a label-free mass spectrometry-based proteomics approach, the composition of the PC is examined for a set of nanotubes (NTs) including unmodified and carboxylated single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), polyvinylpyrrolidone (PVP)-coated MWCNT (MWCNT-PVP), and nanoclay. NTs are incubated for 1 h in simulated cell culture conditions, then washed, resuspended in PBS, and assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for their associated PC. To determine those attributes that influence PC formation, the NTs are extensively characterized. NTs had negative zeta potentials in water (SWCNT-COOH < MWCNT-COOH < unmodified NTs) while carboxylation increases their hydrodynamic sizes. All NTs are also found to associate a common subset of proteins including albumin, titin, and apolipoproteins. SWCNT-COOH and MWCNT-COOH are found to bind the greatest number of proteins (181 and 133 respectively) compared to unmodified NTs (<100), suggesting covalent binding to protein amines. Modified NTs bind a number of unique proteins compared to unmodified NTs, implying hydrogen bonding and electrostatic interactions are involved in PC formation. PVP-coating of MWCNT did not influence PC composition, further reinforcing the possibility of hydrogen bonding and electrostatic interactions. No relationships are found between PC composition and corresponding isoelectric point, hydropathy, or aliphatic index, implying minimal roles of hydrophobic interaction and pi-stacking.

Proteomic study of low-temperature responses in strawberry cultivars (Fragaria x ananassa) that differ in cold tolerance.

To gain insight into the molecular basis contributing to overwintering hardiness, a comprehensive proteomic analysis comparing crowns of octoploid strawberry (Fragaria × ananassa) cultivars that differ in freezing tolerance was conducted. Four cultivars were examined for freeze tolerance and the most cold-tolerant cultivar ('Jonsok') and least-tolerant cultivar ('Frida') were compared with a goal to reveal how freezing tolerance is achieved in this distinctive overwintering structure and to identify potential cold-tolerance-associated biomarkers. Supported by univariate and multivariate analysis, a total of 63 spots from two-dimensional electrophoresis analysis and 135 proteins from label-free quantitative proteomics were identified as significantly differentially expressed in crown tissue from the two strawberry cultivars exposed to 0-, 2-, and 42-d cold treatment. Proteins identified as cold-tolerance-associated included molecular chaperones, antioxidants/detoxifying enzymes, metabolic enzymes, pathogenesis-related proteins, and flavonoid pathway proteins. A number of proteins were newly identified as associated with cold tolerance. Distinctive mechanisms for cold tolerance were characterized for two cultivars. In particular, the 'Frida' cold response emphasized proteins specific to flavonoid biosynthesis, while the more freezing-tolerant 'Jonsok' had a more comprehensive suite of known stress-responsive proteins including those involved in antioxidation, detoxification, and disease resistance. The molecular basis for 'Jonsok'-enhanced cold tolerance can be explained by the constitutive level of a number of proteins that provide a physiological stress-tolerant poise.

Reproducible method to enrich membrane proteins with high purity and high yield for an LC-MS/MS approach in quantitative membrane proteomics.

The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC-MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC-MS/MS label-free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC-MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 µg/10 million HT29-MTX cells. LC-MS/MS analysis of the membrane-enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.

Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture.

Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes.

A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS.

Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs ≤ 30% across 10 technical replicates, and at least 93.6% (up to 2,013) had R(2) ≥ 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification.