Secretome of the preimplantation human embryo by bottom-up label-free proteomics

A bottom-up label-free mass spectrometric proteomic strategy was used to analyse the protein profiles of the human embryonic secretome. Culture media samples used for embryonic culture of patients undergoing intracytoplasmic sperm injection cycles were selected as a test case for this exploratory proof-of-principle study. The media were stored after embryo transfer and then pooled into positive (n = 8) and negative (n = 8) implantation groups. The absolute quantitative bottom-up technique employed a multidimensional protein identification technology based on separation by nano-ultra-high pressure chromatography and identification via tandem nano-electrospray ionization mass spectrometry with data-independent scanning in a hydrid QqTOF mass spectrometer. By applying quantitative bottom-up proteomics, unique proteins were found exclusively in both the positive- and negative-implantation groups, which suggest that competent embryos express and secrete unique biomarker proteins into the surrounding culture medium. The selective monitoring of these possible secretome biomarkers could make viable procedures using single-embryo transfer.     

Label-free LC-MS/MS quantitative proteomics for large-scale biomarker discovery in complex samples

Proteomic platforms that enable researchers to profile a high number of proteins across large sets of complex samples hold a great potential for biomarker discovery. LC-MS/MS-based methods can be used to analyse many samples without the need for protein labelling. As the analysis is a sequential process, the performance of the system has to be consistent throughout the entire experiment. In this study we used a set of spiked serum samples as well as a set of 55 clinical serum samples from schizophrenia patients and healthy volunteers to show that the label-free proteomic approach yields reproducible results across a large number of samples and can be used to accurately measure the relative protein abundance. Using this approach, we identified 1709 serum proteins covering a dynamic range of over three orders of magnitude. We believe that label-free quantitative proteomics is especially suited for biomarker discovery in large sample sets.     

Multiwalled carbon nanotubes modified with 2-aminobenzothiazole modified for uniquely selective solid-phase extraction and determination of Pb(II) ion in water samples

A solid phase extraction method is presented for the selective preconcentration and/or separation of trace Pb(II) on multiwalled carbon nanotubes modified with 2-aminobenzothiazole. Inductively coupled plasma optical emission spectrometry was used for detection. The effects of pH, shaking time, sample flow rate and volume, elution condition and interfering ions were examined using batch and column procedures. An enrichment factor of 100 was accomplished. Common other ions do not interfere in both the separation and determination. The maximum adsorption capacity of the sorbent at optimum conditions is 60.3?mg?g^?1 of Pb(II), the detection limit (3??) is 0.27?ng?mL^?1, and the relative standard deviation is 1.6% (n?=?8). The method was validated using a certified reference material, and has been applied to the determination of trace Pb(II) in water samples with satisfactory results.

Improvement of precision for pipetting blood serum samples into a graphite furnace

Graphite furnace atomic absorption spectrometry is a well-established technique for trace metal determination in blood and serum samples. For this kind of samples, a R.S.D. of up to 10% is considered acceptable, especially for those elements, the concentrations of which do not allow high dilution. Among the reasons that contribute to an increase of the S.D. is the retention of proteins (and analyte) in the sample dispenser capillary, because proteins might be adsorbed on polymeric surfaces. In the present work, the interaction protein/polymer was studied, considering the amount of protein that could be retained by the capillary, the influence of sample dilution, time of contact and the possible co-adsorption of metals. Serum proteins were retained in the capillary, depending on the material (Tygon>silicone rubber>poly(tetrafluorethylene)); dilution did not prevent the adsorption, and only 30 s of contact were enough for the adsorption to occur. Using a column filled with PTFE powder (60 mesh), it was possible to observe that metals were co-adsorbed to a large extent. Water, diluted nitric acid or aqueous solutions of Triton X-100 were not able to promote the complete desorption of the proteins retained by the polymeric materials. Total elution was achieved with methanol, and its use as rinse solution decreased the R.S.D. (n=10) for aluminium, manganese and chromium determination in serum to <10%.     

Comparing Efficiency of Lysis Buffer Solutions and Sample Preparation Methods for Liquid Chromatography–Mass Spectrometry Analysis of Human Cells and Plasma

The use of a proper sample processing methodology for maximum proteome coverage and high-quality quantitative data is an important choice to make before initiating a liquid chromatography–mass spectrometry (LC–MS)-based proteomics study. Popular sample processing workflows for proteomics involve in-solution proteome digestion and single-pot, solid-phase-enhanced sample preparation (SP3). We tested them on both HeLa cells and human plasma samples, using lysis buffers containing SDS, or guanidinium hydrochloride. We also studied the effect of using commercially available depletion mini spin columns before SP3, to increase proteome coverage in human plasma samples. Our results show that the SP3 protocol, using either buffer, achieves the highest number of quantified proteins in both the HeLa cells and plasma samples. Moreover, the use of depletion mini spin columns before SP3 results in a two-fold increase of quantified plasma proteins. With additional fractionation, we quantified nearly 1400 proteins, and examined lower-abundance proteins involved in neurodegenerative pathways and mitochondrial metabolism. Therefore, we recommend the use of the SP3 methodology for biological sample processing, including those after depletion of high-abundance plasma proteins.     

Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma

The domestic chicken (Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2 and 7 years of age. An experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality (Harris, Clin Chem 20: 1535–1542, 1974). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CV_W) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered “healthy” and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Analytical variability (CV_A) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CV_W in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase (p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation.     

Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for Label-Free Single-Cell Proteomics**

We combined efficient sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in rapid label-free analyses. WWA employs large isolation windows to intentionally co-isolate and co-fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2-identified proteins by ≈40 % relative to standard data-dependent acquisition. For a 40-min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single-cell-sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up- or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.     

Two-step elution of human serum proteins from different glass-modified bioactive surfaces: a comparative proteomic analysis of adsorption patterns

Plasma protein adsorption patterns on surfaces may give vital information to evaluate biocompatibility of biomaterials designed for direct blood-contacting applications or tissue integration. Adsorption of human serum proteins on four different types of biomaterials (glass, aminosilanized glass, hyaluronan and sulfated hyaluronan) was analyzed by two-dimensional electrophoresis. Desorption of proteins from the surfaces was first classically achieved by sodium dodecyl sulfate (SDS) elution. We introduced a second elution step (by use of isoelectric focusing (IEF) sample buffer consisting of urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propansulfonate, and dithioerythritol) which allows more stringent elution conditions and is a tool to evaluate the protein adsorption strength to biomaterials. Moreover, the two-step elution may discriminate between irreversible and reversible adsorption of plasma proteins for biomaterials, thus helping to elucidate the structure of protein multilayers which form a complex system at the surfaces. The IEF sample buffer proved not to alter the biomaterial structure and integrity. Hydrophobic bonds resulted to be the main strength driving protein adsorption onto our biomaterials. Apolipoproteins were the most important proteins interacting with the surfaces suggesting that high-density lipoprotein (HDL) particles could play a role in biocompatibility due to their beneficial effects on endothelial cells.     

High-performance liquid chromatography of proteins using chemically-modified silica supports

Summary Highly efficient and fast exclusion-chromatographic separations of proteins are possible on chemically-modified, silica stationary phases. By optimizing the pH and the ionic strength of the aqueous eluent secondary interactions of the samples with surface groups can be excluded. Bonded propylamide groups proved to possess optimum properties for exclusion chromatography. With other functional groups adsorption effects cannot be excluded totally. The optimum pore size distribution for protein separation up to relative molecular masses of 500,000 daltnons is between 10nm and 50nm. With these silica-based phases the pore size distribution, the pore volume and the packing characteristics are independent of the eluent, therefore the same column can be used with aqueous as well with organic eluents. It is possible to correlate the elution volume (molecular size) of proteins with those of polystyrene standars. The recovery of the proteins and their biological activity has always been better than 90%. The potentialities of adsorption chromatography of proteins on chemically-bonded stationary plases with different functional groups are demonstrated.     

Tracking Pathogen Infections by Time-Resolved Chemical Proteomics

Studying the dynamic interaction between host cells and pathogen is vital but remains technically challenging. We describe herein a time-resolved chemical proteomics strategy enabling host and pathogen temporal interaction profiling (HAPTIP) for tracking the entry of a pathogen into the host cell. A novel multifunctional chemical proteomics probe was introduced to label living bacteria followed by in vivo crosslinking of bacteria proteins to their interacting host-cell proteins at different time points initiated by UV for label-free quantitative proteomics analysis. We observed over 400 specific interacting proteins crosslinked with the probe during the formation of Salmonella-containing vacuole (SCV). This novel chemical proteomics approach provides a temporal interaction profile of host and pathogen in high throughput and would facilitate better understanding of the infection process at the molecular level.     

Identification evidence unraveled by strict proteomics rules toward forensic samples

Snake venom is a complex mixture of proteins and peptides secreted by venomous snakes from their poison glands. Although proteomics for snake venom composition, interspecific differences, and developmental evolution has been developed for a decade, current diagnosis or identification techniques of snake venom in clinical intoxication and forensic science applications are mainly dependent on morphological and immunoassay. It could be expected that the proteomics techniques directly offer great help. This work applied a bottom-up proteomics method to identify proteins’ types and species attribution in suspected snake venom samples using ultrahigh-performance liquid chromatography–quadrupole-electrostatic field Orbitrap tandem mass spectrometric technique, and cytotoxicity assay was amended to provide a direct evidence of toxicity. Toward the suspicious samples seized in the security control, sample pretreatment (in-sol and in-gel digestion) and data acquisition (nontargeted and targeted screening) modes complemented and validated each other. We have implemented two consequent approaches in identifying the species source of proteins in the samples via the points of venom proteomics and strict forensic identification. First, we completed a workflow consisting of a proteomics database match toward an entire SWISS-PROT (date 2018-11-22) database and a result-directed specific taxonomy database. The latter was a helpful hint to compare master protein kinds and reveal the insufficiency of specific venom proteomics characterization rules. Second, we suggested strict rules for protein identification to meet the requirements of forensic science on improved identification correctness, that is, (1) peptide spectrum matches confidence, peptide confidence, and protein confidence were both high (with the false-discovery ratio less than 1%); (2) the number of unique peptides was more than or equal to two in one protein, and (3) within unique peptides, which at least 75% of the ∆m/z of the matched y and b ions were less than 5 ppm. We identified these samples as cobra venom containing 10 highly abundant proteins (P00597, P82463, P60770, Q9YGI4, P62375, P49123, P80245, P60302, P01442, and P60304) from two snake venom protein families (acid phospholipase A2 and three-finger toxins), and the most abundant proteins were cytotoxins.     

Preparation of magnetic molecularly imprinted polymer nanoparticles by surface imprinting by a sol–gel process for the selective and rapid removal of di‐(2‐ethylhexyl) phthalate from aqueous solution

Magnetic molecularly imprinted polymer nanoparticles for di‐(2‐ethylhexyl) phthalate were synthesized by surface imprinting technology with a sol–gel process and used for the selective and rapid adsorption and removal of di‐(2‐ethylhexyl) phthalate from aqueous solution. The prepared magnetic molecularly imprinted polymer nanoparticles were characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and vibrating sample magnetometry. The adsorption of di‐(2‐ethylhexyl) phthalate onto the magnetic molecularly imprinted polymer was spontaneous and endothermic. The adsorption equilibrium was achieved within 1 h, the maximum adsorption capacity was 30.7 mg/g, and the adsorption process could be well described by Langmuir isotherm model and pseudo‐second‐order kinetic model. The magnetic molecularly imprinted polymer displayed a good adsorption selectivity for di‐(2‐ethylhexyl) phthalate with respect to dibutyl phthalate and di‐n‐octyl phthalate. The reusability of magnetic molecularly imprinted polymer was demonstrated for at least eight repeated cycles without significant loss in adsorption capacity. The adsorption efficiencies of the magnetic molecularly imprinted polymer toward di‐(2‐ethylhexyl) phthalate in real water samples were in the range of 98–100%. These results indicated that the prepared adsorbent could be used as an efficient and cost‐effective material for the removal of di‐(2‐ethylhexyl) phthalate from environmental water samples.     

Selective solid-phase extraction of trace thallium with nano-Al<Subscript>2</Subscript>O<Subscript>3</Subscript> from environmental samples

A novel sorbent, nano-Al₂O₃ was employed for the separation and preconcentration of thallium from aqueous solution in batch equilibrium experiments. It was found that the adsorption percentage of thallium ions was close to 100% at pH 4.5, and the desorption by 1.0 mL of 0.25 M HCl reached 99%. The adsorption equilibrium was well described by the Langmuir isotherm model with maximum adsorption capacity of 5.78 mg/g (20 ± 0.1°C). The enrichment factor values of Tl(III) was 25 for 25 mL sample. Detection limit of thallium (3σ, n = 11) equal to 0.8 μg/mL and relative standard deviation (2.4%) were obtained. The method has been successfully applied to the determination of trace thallium in some environmental samples and the certified reference material polymetallic nodule (GBW07296) with satisfactory results.     

Digital Microfluidics and Magnetic Bead-Based Intact Proteoform Elution for Quantitative Top-down Nanoproteomics of Single C. elegans Nematodes

While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up proteomics workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40 % formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analyses of single Caenorhabditis elegans nematodes, thereby increasing the number of proteoform identifications compared to in-tube sample preparation by 46 %. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in the abundance of proteoforms not distinguishable by bottom-up proteomics. The presented workflow will facilitate proteoform-directed analysis on samples of limited availability.     

Preconcentration of trace amounts of cadmium in aqueous samples on minicolumns packed with Chromosorb series gas chromatographic stationary phases and its determination by electrothermal atomic absorption spectrometry.

The adsorption characteristics of five commercially available Chromosorb GC stationary phases towards Cd2+ and their application to the preconcentration and ETAAS determination of Cd2+ in several water samples were studied. The experimental results indicated that although all of the five Chromosorb GC stationary phases studied can retain Cd2+ quantitatively from aqueous solutions at appropriate pH values without adding chelating reagent. Chromosorb 105 displayed the highest adsorption capacity. A highly sensitive, simple methodology for preconcentration and ETAAS determination of trace amounts of cadmium in natural water samples using a Chromosorb 105 packed minicolumn is proposed. Conditions for quantitative and reproducible preconcentration, elution and subsequent ETAAS determination were established. The high retention efficiency (> 95%) for Cd2+ provides a sensitivity enhancement of 100-fold for a 200 mL sample volume with a detection limit of 6.2 ng L(-1) (3 sigma).     

Bifunctionalized SBA-15 as a novel micropipette tip sorbent for selective removal and enrichment of biomolecules

Selective enrichment or removal of specific proteins prior to analysis can minimize nonspecific interferences as well as information loss, which has been an important issue in current proteomics fields. In this work, two kinds of mesoporous silica SBA-15 (mean pore diameter of 5 nm and 7 nm), bifunctionalized with quaternary ammonium and C18 groups via silylanization reaction, was used to investigate the adsorption behavior for different proteins (bovine serum albumin (BSA), ovalbumin (OVA), hemoglobin (Hb), lysozyme (Lys) and cytochrome c (cyt c)). As possessing anion exchange (quaternary ammonium) groups, the bifunctionalized SBA-15 showed selective adsorption of the negative charged proteins, BSA and OVA. Based on these properties, sequential fractionation based on different SBA-15 materials as micropipette tip sorbents was developed for sample preparation prior to protein analysis. As expected, after the sequential treatment of the sample, the detection signal of the high abundant BSA was significantly decreased and that of the low abundant cyt c was obviously enlarged, which solved the problem that low abundant protein was suppressed by adjacent high abundant protein. The sample preparation technique significantly improved protein identification and this sequential fractionation approach could be potentially applied to extend information on the protein identification for biological samples with a wide dynamic range.     

Preconcentration of trace aluminum (III) ion using a nanometer-sized TiO2-silica composite modified with 4-aminophenylarsonic acid, and its determination by ICP-OES

We describe a nanometer sized composite material made from titanium dioxide and silica that was chemically modified with 4-aminophenylarsonic acid and used for selective solid-phase extraction, separation and preconcentration of of aluminum(III) prior to its determination by ICP-OES. Under optimized conditions, the static adsorption capacity is 56.58?mg?g^?1, the enrichment factor is 150, the relative standard deviation is 1.6% (for n?=?11), and the detection limit (3?s) is 60?pg?mL^?1. The method was validated by analyzing the reference materials GBW 09101 (hair) and GBW 10024 (scallop) and successfully applied to the determination of trace Al(III) in spiked water samples and human urine, with recoveries ranging from 96% to 101%.

氧化石墨烯/壳聚糖微球吸附-电感耦合等离子体质谱法测定地质样品中痕量钽EI北大核心CSCD

建立了氧化石墨烯/壳聚糖微球吸附-电感耦合等离子体质谱法测定地质样品中痕量Ta的方法。合成了氧化石墨烯/壳聚糖微球,将其应用于地质样品中痕量Ta的分离富集,采用电感耦合等离子体质谱对富集后的试液进行测定。对氧化石墨烯含量、配体种类和浓度、pH、吸附时间、吸附剂用量等吸附条件和洗脱剂类型、体积、洗脱时间等洗脱条件进行了优化,在优化的条件下,对地质样品标准物质进行测定,Ta的方法检出限为2.0 ng/g,富集倍数为26.7倍。     

Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole--time-of-flight mass spectrometer

The application of microfabricated devices coupled to a quadrupole time-of-flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub-nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several microL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post-separation channel. Rapid identification of trace level peptides is also demonstrated using on-line tandem mass spectrometry and database searching with peptide sequence tags.     

Sensitive determination of polycyclic aromatic hydrocarbons in water samples by HPLC coupled with SPE based on graphene functionalized with triethoxysilane

The graphene functionalized with (3‐aminopropyl) triethoxysilane was synthesized by a simple hydrothermal reaction and applied as SPE sorbents to extract trace polycyclic aromatic hydrocarbons (PAHs) from environmental water samples. These sorbents possess high adsorption capacity and extraction efficiency due to strong adsorption ability of carbon materials and large specific surface area of nanoparticles, and only 10 mg of sorbents are required to extract PAHs from 100 mL water samples. Several condition parameters, such as eluent and its volume, adsorbent amount, sample volume, sample pH, and sample flow rate, were optimized to achieve good sensitivity and precision. Under the optimized extraction conditions, the method showed good linearity in the range of 1–100 μg/L, repeatability of the extraction (the RSDs were between 1.8 and 2.9%, n = 6), and satisfactory detection limits of 0.029–0.1 μg/L. The recoveries of PAHs spiked in environmental water samples ranged from 84.6 to 109.5%. All these results demonstrated that this new SPE technique was a viable alternative to conventional enrichment techniques for the extraction and analysis of PAHs in complex samples.