Nano-electrospray Tandem Mass Spectrometry Research Articles

Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma.

Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.

Characterization of Triacylglycerol Estolide Isomers Using High-Resolution Tandem Mass Spectrometry with Nanoelectrospray Ionization

Triacylglycerol estolides (TG-EST) are biologically active lipids extensively studied for their anti-inflammatory and anti-diabetic properties. In this work, eight standards of TG-EST were synthesized and systematically investigated by nanoelectrospray tandem mass spectrometry. Mass spectra of synthetic TG-EST were studied with the purpose of enabling the unambiguous identification of these lipids in biological samples. TG-EST glycerol sn-regioisomers and isomers with the fatty acid ester of hydroxy fatty acid (FAHFA) subunit branched in the ω-, α-, or 10-position were used. Ammonium, lithium, and sodium adducts of TG-EST formed by nanoelectrospray ionization were subjected to collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD). Product ion spectra allowed for identification of fatty acid (FA) and FAHFA subunits originally linked to the glycerol backbone and distinguished the α-branching site of the FAHFA from other estolide-branching isomers. The ω- and 10-branching sites were determined by combining CID with ozone-induced dissociation (OzID). Lithium adducts provided the most informative product ions, enabling characterization of FA, hydroxy fatty acid (HFA), and FAHFA subunits. Glycerol sn-regioisomers were distinguished based on the relative abundance of product ions and unambiguously identified using CID/OzID of lithium and sodium adducts.

Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.

The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other. In addition, alkaline-pH-MS/MS showed identification capacity for a higher proportion of peptides with negative grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled enrichment preference toward histidine-, lysine-, methionine-, and proline-containing peptides. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS method outperformed the conventional 120 min low-pH-MS/MS method in both the identification of amino acid variants and protein groups. Therefore, we established the alkaline-pH-MS/MS method as a simple, competitive, alternative method to low-pH-MS/MS for global proteomic analysis.

Overview of Adductomics in Toxicology.

Adductomics is epidemiology at the molecular level. Untargeted adductomics compares levels of chemical adducts on albumin, hemoglobin, and DNA between healthy and exposed individuals. The goal is to determine a cause-and-effect relationship between chemical exposure and illness. Chemical exposures are not necessarily due to synthetic chemicals but are often due to oxidation products of naturally occurring lipids, for example, 4-hydroxynonenal and acrolein produced by lipid peroxidation of arachidonic and linoleic acids. The preferred method used in adductomics is ultra-high pressure liquid chromatography coupled to with nanoelectrospray tandem mass spectrometry. The mass of the adduct indicates its structure and identifies the chemical. The advantages of molecular epidemiology include information about the many toxicants to which a person is exposed over a period of weeks or months and the relative exposure levels. The disadvantage is the absence of information about the mechanism of toxicity. Untargeted adductomics examines albumin and hemoglobin adducts, which serve as biomarkers of exposure but do not identify the proteins and genes responsible for the toxicity. Targeted adductomics is used when the origin of the toxicity is known. This can be either an adducted protein, such as the butyrylcholinesterase protein modified by nerve agents, or a toxicant, such as acetaminophen. Untargeted adductomics methods have identified potential protein adduct biomarkers of breast cancer, colorectal cancer, childhood leukemia, and lung cancer. Adductomics is a new research area that offers structural insights into chemical exposures and a platform for the discovery of disease biomarkers. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

LEDGF is a new growth factor in fetal serum

Fetal serum supports the immortal growth of mammalian cell lines in culture while adult serum leads to the terminal differentiation and death of cells in culture. Many of the proteins in fetal serum that support the indefinite division and growth of cancerous cell lines remain obscure. The peptides and proteins of fetal versus adult serum were analyzed by liquid chromatography, nano electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Three batches of fetal serum contained the Alpha Fetoprotein marker while adult serum batches did not. Insulin (INS), and insulin-like growth factor (ILGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF) were increased in fetal serum. New fetal growth factors including MEGF, HDGFRP and PSIP1 and soluble growth receptors such as TNFR, EGFR, NTRK2 and THRA were discovered. Addition of insulin or the homeotic transcription factor PSIP1, also referred to as Lens Epithelium Derived Growth Factor (LEDGF), partially restored the rounded phenotype of rapidly dividing cells but was not as effective as fetal serum. Thus, a new growth factor in fetal serum, LEDGF/PSIP1, was directly observed by tandem mass spectrometry and confirmed by add back experiments to cell culture media alongside insulin.

Rapid identification of bear bile powder from other bile sources using chip-based nano-electrospray ionization tandem mass spectrometry.

Bear bile powder (BBP) is a widely used traditional Chinese medicine (TCM), and bile acids (BAs) are the main active components in BBP. Due to the scarcity of BBP resources, adulterations often occur in the market. Conventional methods to distinguish them are usually complicated and time-consuming. To enhance effectiveness and accuracy, a rapid and rough analytical method is desperately needed. In this study, a rapid strategy using chip-based nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was established to distinguish BBP from other sources of bile powder (BP). In addition, the results were further verified by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry (UPLC/MS). The precision of the chip-based nano-ESI-MS/MS method was validated to be acceptable with relative standard deviation (RSD) <15%. The distinction between BBP and other sources of BP, including common adulterants of pig bile powder (PBP), cattle bile powder (CBP), sheep bile powder (SBP), and chicken bile powder (CkBP), can be observed in the spectra. By using orthogonal partial least-squares discriminant analysis (OPLS-DA), more potential m/z markers were investigated. A BAs-related m/z marker of 498.3 was discovered as a typical differential molecular ion peak and was identified as tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) in BBP. The proposed strategy has simple sample pretreatment steps and significantly shortened analysis time. As an emerging technology, chip-based nano-ESI-MS not only provides a reference for the rapid distinction of adulterated Chinese medicines, but also provides some insights into the identification of other chemicals and foods.

Top-down and Bottom-up Approaches Revealed New Categories of Peptides from the Venom of Moroccan Scorpion Androctonus mauretanicus

Background: Androctonus mauretanicus (Am ) is one of the most hazardous scorpions in Morocco and has a highly toxic venom responsible for severe cases of envenomation. However, few studies have focused on deciphering its proteic composition. Objectives: Herein, we aim to map out the complete proteome of the Am venom filtrate to highlight its complexity and the polymorphism of its toxic content. This, in turn, will lead to a deeper understanding of the toxins’ mechanism of action and will help uncover those with therapeutic potential. Methods: Top-down and bottom-up proteomic approaches were used complementarily to decipher the proteome of the Am venom. These approaches were carried out on nano-high liquid chromatography coupled to nano-electrospray tandem mass spectrometry (Nano-LC-ESI-MS/MS). Results: Am venom encloses a complex mixture of 269 different compounds with molecular weights ranging from 1618.74 to 14 214.84 Da. The most abundant ones showed masses from 6185.92 to 7899.53 Da (53.89%) followed by those ranging from 2079.25 to 5969.63 Da (37.81%). Interestingly, the combination of the results of both approaches allowed the screening of a total of 112 peptides. The highest percentage was represented by neuropeptides (87%), including NaTxs, KTxs, ClTxs, venom proteins, venom neuropeptides, and myotropic neuropeptides. Moreover, other peptides were identified, such as antimicrobial peptides, amphipathic peptides, cysteine-rich venom peptides, enzymes, kunitz-type inhibitors and orphan peptides. Conclusion: The Am venom appears to contain a great number of diverse peptides, some of which could prospectively be exploited for their pharmaceutical potential.

Dilute-and-shoot-based direct nano-electrospray ionization tandem mass spectrometry as screening methodology for multivitamins in dietary supplement and human urine

IntroductionIn recent years, analytical screening methods for simultaneous detection of multivitamins have gained substantial attention to ensure quality and public confidence in dietary supplements. Even so, few analytical methods have been proposed for simultaneous analysis of multivitamin constituents due to the large divergence in chemical characteristics. ObjectivesIn the present study, the objective was to develop a simple and rapid direct nano-electrospray ionization-tandem mass spectrometry (DI-nano-ESI-MS/MS) method for targeted detection of water soluble vitamins, fat soluble vitamins, amino acids, royal jelly, ginkgo biloba, and ginseng in a dietary supplement. The applicability of dilute-and-shoot-based DI-nano-ESI-MS/MS to analyze the same tested compounds and their related metabolites in clinical samples was also examined. MethodsIntact urine mixed with the ionization solvent was loaded (4-μL aliquot) into a nanospray (NS) capillary of 1-μm tip diameter. The NS capillary was then fitted into an off-line ion source at a distance of 5 mm from MS aperture. The sample was directly injected by applying a voltage of 1.1 kV, producing a numerous of m/z peaks for analysis in mere minutes. ResultsThe DI-nano-ESI-MS/MS method successfully identified almost all dietary supplement components, as well as a plethora of component-related metabolites in clinical samples. In addition, a new merit of the proposed method for the detection of index marker and chemical contaminants as well as subspecies identification was investigated for further quality evaluation of the dietary supplement. ConclusionsThe previous findings illustrated that DI-nano-ESI-MS/MS approach can emerge as a powerful, high throughput, and promising analytical tool for screening and accurate detection of various pharmaceuticals and ingredient in dietary supplements as well as biological fluids.

Blood Proteomics-Based Detection of Upregulated Lipid Metabolism and Immune Dysfunction in an Elite Adventure Athlete Trekking Across Antarctica

Abstract Objectives Proteomics when combined with psychological, nutrition, and performance measures may serve as a useful monitoring system for immune dysfunction, training distress, and exercise-induced muscle damage and exhaustion in athletes. Global proteomics monitoring of an elite adventure athlete (age 33 years) was conducted over a 28-week period that culminated in the successful, unassisted 2-month trek across Antarctica (1500 km). Methods Training distress was monitored weekly using the 19-item, validated Training Distress Scale (TDS). Weekly dried blood spot (DBS) specimens were collected via fingerprick blood drops onto standard blood spot cards. DBS proteins were measured with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) in data-independent acquisition (DIA) mode, and 712 proteins were identified and quantified. Results The participant experienced a decrease of 11.4 kg in body mass during the Antarctica trek. The 28-week period was divided into time segments based on TDS scores, and a contrast analysis between weeks 5–8 (low TDS) and weeks 20–23 (high TDS, last month of Antarctica trek) showed that 31 proteins (n = 20 immune related, n = 14 nutrition related with n = 8 in dual roles) were upregulated and 35 (n = 17 immune related) were downregulated. Protein-protein interaction (PPI) networks and gene ontology (GO) biological process analysis supported an increase in plasma lipoprotein particle remodeling, regulation of lipid transport, retinoid metabolic process, and vitamin transport due to high energy intake (7048 kcal/d). PPI networks also supported a dichotomous immune response. GO terms for the upregulated immune proteins showed an increase in regulation of the immune system process, especially inflammation, complement activation, and leukocyte mediated immunity. GO terms for the downregulated immune-related proteins indicated a decrease in several aspects of the overall immune system process including neutrophil degranulation and the antimicrobial humoral response. Conclusions These proteomics data support a dysfunctional immune response in an elite adventure athlete during a sustained period of mental and physical distress, high energy intake, and significant loss of body mass while trekking solo across Antarctica. Funding Sources Standard Process, Inc., Palmyra, WI.

Proteomics-Based Detection of Immune Dysfunction in an Elite Adventure Athlete Trekking Across the Antarctica.

Proteomics monitoring of an elite adventure athlete (age 33 years) was conducted over a 28-week period that culminated in the successful, solo, unassisted, and unsupported two month trek across the Antarctica (1500 km). Training distress was monitored weekly using a 19-item, validated training distress scale (TDS). Weekly dried blood spot (DBS) specimens were collected via fingerprick blood drops onto standard blood spot cards. DBS proteins were measured with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) in data-independent acquisition (DIA) mode, and 712 proteins were identified and quantified. The 28-week period was divided into time segments based on TDS scores, and a contrast analysis between weeks five and eight (low TDS) and between weeks 20 and 23 (high TDS, last month of Antarctica trek) showed that 31 proteins (n = 20 immune related) were upregulated and 35 (n = 17 immune related) were downregulated. Protein–protein interaction (PPI) networks supported a dichotomous immune response. Gene ontology (GO) biological process terms for the upregulated immune proteins showed an increase in regulation of the immune system process, especially inflammation, complement activation, and leukocyte mediated immunity. At the same time, GO terms for the downregulated immune-related proteins indicated a decrease in several aspects of the overall immune system process including neutrophil degranulation and the antimicrobial humoral response. These proteomics data support a dysfunctional immune response in an elite adventure athlete during a sustained period of mental and physical distress while trekking solo across the Antarctica.

Direct nano-electrospray ionization tandem mass spectrometry for the quantification and identification of metronidazole in its dosage form and human urine.

A rapid, sensitive and direct nano-electrospray ionization-tandem mass spectrometry (NS-ESI-MS/MS) method, using an offline nanospray (NS) capillary, has been developed and validated for the analysis of metronidazole (MTZ). A mixture of 2 µl MTZ sample solution prepared in an ionization solvent consisting of methanol : water : formic acid in a ratio of 80 : 20 : 0.3, together with 2 µl of an internal standard (IS), 1,3,6-polytyrosine, is loaded into the back of the NS capillary. The NS capillary was fitted into the ion source at a distance of 3 mm between the NS tip and MS orifice. The sample is then analysed and acquired a sustainable signal that allowed for data compilation across various data points for MTZ identification and quantification. The quantification relied on the ratio of the [M + H]+ peaks of MTZ and IS with m/z values of 172.0717 and 182.0812, respectively, while the identification relied on the MS/MS of the precursor ions [M + H]+ of both compounds and their fragments at 128.05 for MTZ and 165.1 and 136.07 for the IS. The NS-ESI-MS/MS method was accurate and precise for the quantification of MTZ over the concentration range from 2.5 to 25 000 ng ml−1. The applicability of the method was confirmed by MTZ analysis in its pharmaceutical dosage form and detection of the analyte in clinical human urine samples without any sample treatment procedure.

Venom Proteome of Spine-Bellied Sea Snake (Hydrophis curtus) from Penang, Malaysia: Toxicity Correlation, Immunoprofiling and Cross-Neutralization by Sea Snake Antivenom

The venom proteome of Hydrophis curtus (synonym: Lapemis hardwickii) from Penang, Malaysia was investigated with nano-electrospray ionization-liquid chromatography tandem mass spectrometry (ESI-LCMS/MS) of the reverse-phase high-performance liquid chromatography (HPLC) venom fractions. Thirty distinct protein forms were identified as toxins from ten families. The three major protein families were phospholipase A2 (PLA2, 62.0% of total venom proteins), three-finger toxin (3FTX, 26.33%) and cysteine-rich secretory protein (CRiSP, 9.00%). PLA2 comprises diverse homologues (11 forms), predominantly the acidic subtypes (48.26%). 3FTX composed of one short alpha-neurotoxin (SNTX, 22.89%) and four long alpha-neurotoxins (LNTX, 3.44%). Both SNTX and LNTX were lethal in mice (intravenous LD50 = 0.10 and 0.24 μg/g, respectively) but the PLA2 were non-lethal (LD50 >1 μg/g). The more abundant and toxic SNTX appeared to be the main driver of venom lethality (holovenom LD50 = 0.20 μg/g). The heterologous Sea Snake Antivenom (SSAV, Australia) effectively cross-neutralized the venom (normalized potency = 9.35 mg venom neutralized per g antivenom) and the two neurotoxins in vivo, with the LNTX being neutralized more effectively (normalized potency = 3.5 mg toxin/g antivenom) than SNTX (normalized potency = 1.57 mg/g). SSAV immunorecognition was strong toward PLA2 but moderate-to-weak toward the alpha-neurotoxins, indicating that neutralization of the alpha-neurotoxins should be further improved.

The plasma peptides of ovarian cancer

BackgroundIt may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma by using liquid chromatography and tandem mass spectrometry to identify, quantify and compare the peptides cleaved ex vivo from different clinical populations. The endogenous tryptic peptides of ovarian cancer plasma were compared to breast cancer and female cancer normal controls, other diseases with their matched or normal controls, plus ice cold plasma to control for pre-analytical variation.MethodsThe endogenous tryptic peptides or tryptic phospho peptides (i.e. without exogenous digestion) were analyzed from 200 μl of EDTA plasma. The plasma peptides were extracted by a step gradient of organic/water with differential centrifugation, dried, and collected over C18 for analytical HPLC nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous peptides of ovarian cancer were compared to multiple disease and normal samples from different institutions alongside ice cold controls. Peptides were randomly and independently sampled by LC–ESI–MS/MS. Precursor ions from peptides > E4 counts were identified by the SEQUEST and X!TANDEM algorithms, filtered in SQL Server, before testing of frequency counts by Chi Square (χ2), for analysis with the STRING algorithm, and comparison of precursor intensity by ANOVA in the R statistical system with the Tukey-Kramer Honestly Significant Difference (HSD) test.ResultsPeptides and/or phosphopeptides of common plasma proteins such as HPR, HP, HPX, and SERPINA1 showed increased observation frequency and/or precursor intensity in ovarian cancer. Many cellular proteins showed large changes in frequency by Chi Square (χ2 > 60, p < 0.0001) in the ovarian cancer samples such as ZNF91, ZNF254, F13A1, LOC102723511, ZNF253, QSER1, P4HA1, GPC6, LMNB2, PYGB, NBR1, CCNI2, LOC101930455, TRPM5, IGSF1, ITGB1, CHD6, SIRT1, NEFM, SKOR2, SUPT20HL1, PLCE1, CCDC148, CPSF3, MORN3, NMI, XTP11, LOC101927572, SMC5, SEMA6B, LOXL3, SEZ6L2, and DHCR24. The protein gene symbols with large Chi Square values were significantly enriched in proteins that showed a complex set of previously established functional and structural relationships by STRING analysis. Analysis of the frequently observed proteins by ANOVA confirmed increases in mean precursor intensity in ZFN91, TRPM5, SIRT1, CHD6, RIMS1, LOC101930455 (XP_005275896), CCDC37 and GIMAP4 between ovarian cancer versus normal female and other diseases or controls by the Tukey–Kramer HSD test.ConclusionHere we show that separation of endogenous peptides with a step gradient of organic/water and differential centrifugation followed by random and independent sampling by LC–ESI–MS/MS with analysis of peptide frequency and intensity by SQL Server and R revealed significant difference in the ex vivo cleavage of peptides between ovarian cancer and other clinical treatments. There was striking agreement between the proteins discovered from cancer plasma versus previous biomarkers discovered in tumors by genetic or biochemical methods. The results indicate that variation in plasma proteins from ovarian cancer may be directly discovered by LC–ESI–MS/MS that will be a powerful tool for clinical research.

The plasma peptidome

BackgroundIt may be possible to discover new diagnostic or therapeutic peptides or proteins from blood plasma using LC–ESI–MS/MS to identify, quantify and compare the statistical distributions of peptides cleaved ex vivo from plasma samples from different clinical populations.MethodsA systematic method for the organic fractionation of plasma peptides was applied to identify and quantify the endogenous tryptic peptides from human plasma from multiple institutions by C18 HPLC followed nano electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) with a linear quadrupole ion trap. The endogenous tryptic peptides, or tryptic phospho peptides (i.e. without exogenous digestion), were extracted in a mixture of organic solvent and water, dried and collected by preparative C18. The tryptic peptides from 6 institutions with 12 different disease and normal EDTA plasma populations, alongside ice cold controls for pre-analytical variation, were characterized by mass spectrometry. Each patient plasma was precipitated in 90% acetonitrile and the endogenous tryptic peptides extracted by a stepwise gradient of increasing water and then formic acid resulting in 10 sub-fractions. The fractionated peptides were manually collected over preparative C18 and injected for 1508 LC–ESI–MS/MS experiments analyzed in SQL Server R.ResultsPeptides that were cleaved in human plasma by a tryptic activity ex vivo provided convenient and sensitive access to most human proteins in plasma that show differences in the frequency or intensity of proteins observed across populations that may have clinical significance. Combination of step wise organic extraction of 200 μL of plasma with nano electrospray resulted in the confident identification and quantification ~ 14,000 gene symbols by X!TANDEM that is the largest number of blood proteins identified to date and shows that you can monitor the ex vivo proteolysis of most human proteins, including interleukins, from blood. A total of 15,968,550 MS/MS spectra ≥ E4 intensity counts were correlated by the SEQUEST and X!TANDEM algorithms to a federated library of 157,478 protein sequences that were filtered for best charge state (2+ or 3+) and peptide sequence in SQL Server resulting in 1,916,672 distinct best-fit peptide correlations for analysis with the R statistical system. SEQUEST identified some 140,054 protein accessions, or some ~ 26,000 gene symbols, proteins or loci, with at least 5 independent correlations. The X!TANDEM algorithm made at least 5 best fit correlations to more than 14,000 protein gene symbols with p-values and FDR corrected q-values of ~ 0.001 or less. Log10 peptide intensity values showed a Gaussian distribution from E8 to E4 arbitrary counts by quantile plot, and significant variation in average precursor intensity across the disease and controls treatments by ANOVA with means compared by the Tukey–Kramer test. STRING analysis of the top 2000 gene symbols showed a tight association of cellular proteins that were apparently present in the plasma as protein complexes with related cellular components, molecular functions and biological processes.ConclusionsThe random and independent sampling of pre-fractionated blood peptides by LC-ESI-MS/MS with SQL Server-R analysis revealed the largest plasma proteome to date and was a practical method to quantify and compare the frequency or log10 intensity of individual proteins cleaved ex vivo across populations of plasma samples from multiple clinical locations to discover treatment-specific variation using classical statistics suitable for clinical science. It was possible to identify and quantify nearly all human proteins from EDTA plasma and compare the results of thousands of LC–ESI–MS/MS experiments from multiple clinical populations using standard database methods in SQL Server and classical statistical strategies in the R data analysis system.

A Protein Decomplexation Strategy in Snake Venom Proteomics.

Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).

Identification of Poly(ethylene glycol) and Poly(ethylene glycol)-Based Detergents Using Peptide Search Engines

Poly(ethylene glycol) (PEG) is one of the most common polymer contaminations in mass spectrometry (MS) samples. At present, the detection of PEG and other polymers relies largely on manual inspection of raw data, which is laborious and frequently difficult due to sample complexity and retention characteristics of polymer species in reversed-phase chromatography. We developed a new strategy for the automated identification of PEG molecules from tandem mass spectrometry (MS/MS) data using protein identification algorithms in combination with a database containing "PEG-proteins". Through definition of variable modifications, we extend the approach for the identification of commonly used PEG-based detergents. We exemplify the identification of different types of polymers by static nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS) analysis of pure detergent solutions and data analysis using Mascot. Analysis of liquid chromatography-tandem mass spectrometry (LC-MS/MS) runs of a PEG-contaminated sample by Mascot identified 806 PEG spectra originating from four PEG species using a defined set of modifications covering PEG and common PEG-based detergents. Further characterization of the sample for unidentified PEG species using error-tolerant and mass-tolerant searches resulted in identification of 3409 and 3187 PEG-related MS/MS spectra, respectively. We further demonstrate the applicability of the strategy for Protein Pilot and MaxQuant.

Trisaminohexyl isocyanurate, a urinary biomarker of HDI isocyanurate exposure

Biological monitoring of occupational exposure to 1,6-hexamethylene diisocyanate (HDI)-containing spray-paints is limited to analysis of metabolites of HDI monomer although polymeric HDI isocyanurate constitutes the predominant inhalation and skin exposure for workers in the automotive paint industry. A novel method using nanoflow ultra-performance liquid chromatography coupled to nano-electrospray ionization tandem mass spectrometry (nano-UPLC-ESI-MS/MS) was developed to quantify trisaminohexyl isocyanurate (TAHI), a hydrolysis product of HDI isocyanurate, in the urine of spray-painters. Analytical and internal standards were synthesized in-house and weighted linear regression calibration curves were generated using spiked control urine from non-exposed persons (0.06–7.98 μg/L; N = 13; w = x−2; r = 0.998). Urine samples collected from 15 exposed workers (N = 111) were subjected to acid hydrolysis and extracted with dichloromethane, then derivatized with acetic anhydride. The derivatized product, trisacetamidohexyl isocyanurate (TAAHI), was analyzed using nano-UPLC-ESI-MS/MS. The protocol was sensitive and specific for analysis of TAHI in the urine of exposed workers with a method detection limit at 0.03 μg/L. TAHI was detected in 33 of 111 urine samples and in 11 of 15 workers. This biomarker for HDI isocyanurate is critical to determine the relative potency and dose-relationships between the monomer and oligomer exposure on the development of diisocyanate induced health effects in future studies.

Peptides Released from Foremilk and Hindmilk Proteins by Breast Milk Proteases Are Highly Similar.

Human milk contains active proteases that initiate hydrolysis of milk proteins within the mammary gland. Milk expressed at the beginning of feeding is known as foremilk and that at the end of feeding is known as hindmilk. As hindmilk contains higher fat, vitamins A and E, and higher calories than foremilk, feeding only hindmilk initially and reserving foremilk for later are practiced in some neonatal intensive care units. This study investigated the difference in peptide profiles, predicted milk protease activities, and bioactive peptides between foremilk and hindmilk. Bioactive peptides are short fragments of proteins that influence biological processes. Four mothers pumped 10 mL of their foremilk and 10 mL of their hindmilk into iced containers prepared with antiproteases and the samples were immediately frozen. The peptide profile of each sample was analyzed by liquid chromatography nano-electrospray ionization Orbitrap Fusion tandem mass spectrometry. Peptide abundance (sum of ion intensities) and count (number of unique peptide sequences) in each milk sample were determined from this analysis. The specific enzymes that participated in peptide release were predicted based on the amino acids positioned at each cleavage site. Peptide bioactivity was predicted based on homology to a known functional peptide database and two bioactivity prediction algorithms. Hindmilk contained a higher count of peptides than foremilk. The higher number of unique peptide sequences in hindmilk was related to hydrolysis of β-casein, osteopontin, αs1-casein and mucin-1 via plasmin and elastase cleavage, and possible aminopeptidase and carboxypeptidase activities. Though hindmilk contained a greater number of peptides than foremilk, the overall peptide abundance did not differ and most of the total peptide abundance derived from peptide sequences that were present in both milk types. The presence of higher numbers of predicted bioactive peptides in the hindmilk could indicate that the practice of providing hindmilk rather than foremilk to premature infants could positively impact health outcomes; however, as there are few differences in overall peptide abundance, the overall effect is likely limited.

Simultaneous determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in human retinal DNA by liquid chromatography nanoelectrospray-tandem mass spectrometry.

Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Oxidative damage to mitochondrial DNA (mtDNA) in the retinal pigment epithelium (RPE) may play a key role in AMD. Measurement of oxidative DNA lesions such as 8-oxo-2’-deoxyguanosine (8-oxo-dG) and 8-oxo-2’-deoxyadenosine (8-oxo-dA) in diseased RPE could provide important insights into the mechanism of AMD development. We have developed a liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry method for simultaneous analysis of 8-oxo-dG and 8-oxo-dA in human retinal DNA. The developed method was applied to the analysis of retinal DNA from 5 donors with AMD and 5 control donors without AMD. In mtDNA, the levels of 8-oxo-dG in controls and AMD donors averaged 170 and 188, and 8-oxo-dA averaged 11 and 17 adducts per 106 bases, respectively. In nuclear DNA, the levels of 8-oxo-dG in controls and AMD donors averaged 0.54 and 0.96, and 8-oxo-dA averaged 0.04 and 0.05 adducts per 106 bases, respectively. This highly sensitive method allows for the measurement of both adducts in very small amounts of DNA and can be used in future studies investigating the pathophysiological role of 8-oxo-dG and 8-oxo-dA in AMD and other oxidative damage-related diseases in humans.

Comparative analyses of secreted proteins from the phytopathogenic fungus Verticillium dahliae in response to nitrogen starvation

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC–nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron–proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.