Mammalian plasma membrane proteins as potential biomarkers and drug targets

Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented.     

Surface plasmon resonance-enabled mass spectrometry arrays

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.     

Identification of Peptide tumor markers in a tumor graft model in immunodeficient mice

The medical demand for useful biomarkers is large and still increasing. This is especially true for cancer, because for this disease adequate diagnostic markers with high specificity and sensitivity are still lacking. Despite advances in imaging technologies for early detection of cancer, peptidomic multiplex techniques evolved in recent years will provide new opportunities for detection of low molecular weight (LMW) proteome biomarker (peptides) by mass spectrometry. Improvements in peptidomics research were made based on separation of peptides and/or proteins by their physico-chemical properties in combination with mass spectrometric detection, respectively identification, and sophisticated bioinformatic tools for data analysis. To evaluate the potential of serological tumor marker detection by differential peptide display (DPD) we analyzed plasma samples from a tumor graft model. After subcutaneous injection of HCT-116 cells in immunodeficient mice and their growth to a palpable tumor, plasma samples were analyzed by DPD. The comparison of obtained mass spectrometric data allows discovery of tumor specific peptides which fit well into the biological context of cancer pathogenesis and show a strong correlation to tumor growth. The identified peptides indicate events associated with hyper-proliferation and dedifferentiation of cells from an epithelial origin, which are typical characteristics of human carcinomas. We conclude that these findings are a "proof of principle" to detect differentially expressed, tumor-related peptides in plasma of tumor-bearing mice.     

Precursor ion scans for the targeted detection of stable‐isotope‐labeled peptides

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large‐scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse‐labeled with a stable isotope by metabolic incorporation of ‘heavy’ amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple‐quadrupole ion trap mass spectrometer. PIS‐based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several‐fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS‐based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique. Copyright © 2009 John Wiley & Sons, Ltd.     

Comparison of three methods for fractionation and enrichment of low molecular weight proteins for SELDI-TOF-MS differential analysis

In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers.In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied.The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles.     

Detection of secondary biomarker of met-eGH as a strategy to screen for somatotropin misuse in horseracing

Since the Australian commercialisation of the recombinant equine growth hormone (reGH) in 1998 (EquiGen-5), Bresagen), this reGH, which differs only from eGH by an additional methionine at the N-terminal end (met-eGH), is worldwide suspected to be administered to racehorses as a doping agent. Indeed, the use of this biological drug is considered as a threat to horseracing since it acts both on growth, development or reproductive functions, and on the improvement of performances. In this work, we describe two reliable techniques based on surface plasmon resonance biosensor immunoassay (SPR-BIA) and solid-phase enzyme-linked immunosorbent assay (ELISA) as new, rapid and efficient long-term screening methods applicable to horseracing antidoping analysis. The ELISA and SPR-BIA tests were applied to octanoic acid purified IgGs from serum/plasma samples collected on two thoroughbreds treated with recombinant equine growth hormone for a period of two weeks. The first kinetic study of serum/plasma antibodies raised as a consequence of recombinant equine growth hormone administrations, which allows the detection from eight days up to 200 days after the beginning of the treatment, was performed. In order to trace the occurrence of anti-reGH antibodies in routine analysis and to monitor the animal level exposure to this forbidden molecule, a random population study was conducted on 233 post-race horses.     

Use of NMR metabolomic plasma profiling methodologies to identify illicit growth-promoting administrations

Detection of growth-promoter use in animal production systems still proves to be an analytical challenge despite years of activity in the field. This study reports on the capability of NMR metabolomic profiling techniques to discriminate between plasma samples obtained from cattle treated with different groups of growth-promoting hormones (dexamethasone, prednisolone, oestradiol) based on recorded metabolite profiles. Two methods of NMR analysis were investigated—a Carr–Purcell–Meiboom–Gill (CPMG)-pulse sequence technique and a conventional ¹H NMR method using pre-extracted plasma. Using the CPMG method, 17 distinct metabolites could be identified from the spectra. ¹H NMR analysis of extracted plasma facilitated identification of 23 metabolites—six more than the alternative method and all within the aromatic region. Multivariate statistical analysis of acquired data from both forms of NMR analysis separated the plasma metabolite profiles into distinct sample cluster sets representative of the different animal study groups. Samples from both sets of corticosteroid-treated animals—dexamethasone and prednisolone—were found to be clustered relatively closely and had similar alterations to identified metabolite panels. Distinctive metabolite profiles, different from those observed within plasma from corticosteroid-treated animal plasma, were observed in oestradiol-treated animals and samples from these animals formed a cluster spatially isolated from control animal plasma samples. These findings suggest the potential use of NMR methodologies of plasma metabolite analysis as a high-throughput screening technique to aid detection of growth promoter use.     

Dynamics of Arabidopsis thaliana soluble proteome in response to different nutrient culture conditions

In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.     

Mapping the human plasma proteome by SCX-LC-IMS-MS

The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37,842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

An overview on enrichment methods for cell surface proteome profiling

Cell surface proteins are essential for many important biological processes, including cell–cell interactions, signal transduction, and molecular transportation. With the characteristics of low abundance, high hydrophobicity, and high heterogeneity, it is difficult to get a comprehensive view of cell surface proteome by direct analysis. Thus, it is important to selectively enrich the cell surface proteins before liquid chromatography with mass spectrometry analysis. In recent years, a variety of enrichment methods have been developed. Based on the separation mechanism, these methods could be mainly classified into three types. The first type is based on their difference in the physicochemical property, such as size, density, charge, and hydrophobicity. The second one is based on the bimolecular affinity interaction with lectin or antibody. And the third type is based on the chemical covalent coupling to free side groups of surface‐exposed proteins or carbohydrate chains, such as primary amines, carboxyl groups, glycan side chains. In addition, metabolic labeling and enzymatic reaction‐based methods have also been employed to selectively isolate cell surface proteins. In this review, we will provide a comprehensive overview of the enrichment methods for cell surface proteome profiling.     

血清和血清外泌体的蛋白质组分析及其在肝内胆管癌中的应用

外泌体是由各种类型细胞在正常或非正常生理情况下分泌释放至细胞外且携带多种生物活性分子的细胞外囊泡,在细胞间通讯和免疫应答等生物过程中发挥着重要作用。肝内胆管癌是一种胆道上皮恶性肿瘤,早期无明显临床症状且生存率较低,目前常用的诊断手段包括依赖于影像设备的诊断方式和灵敏度及特异性较低的诊断标志物等,这些手段的不足对发展新的特异性标志物提出了需求。该文对血清中的外泌体进行了分离和表征,并采用液相色谱-质谱技术针对健康组与肝内胆管癌患者组的血清样本和血清外泌体样本进行了无标记定量蛋白质组学分析,分别从两种类型样本中鉴定并筛选到271和430种可信蛋白质。基于血清样本和血清外泌体样本的可信蛋白质定量表达值进行多维统计分析都能将健康组与肝内胆管癌患者组良好地区分开。对血清样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有15个上调和8个下调蛋白质;对血清外泌体样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有33个上调和18个下调蛋白质;基于两种样本筛选到的差异蛋白质中仅有4个是重复的,且基于血清外泌体样本的51个差异蛋白质中有35个蛋白质属于外泌体蛋白质数据库。针对差异蛋白质进行生物学信息分析,与差异蛋白质相关的分子功能、生物过程和信号通路主要涉及天然免疫反应、炎症反应和凝血等过程。该研究为发现肝内胆管癌的潜在生物标志物和探究肝内胆管癌的发生、发展和转移等过程提供了参考和借鉴价值。此外,通过比较研究发现血清外泌体样本能够获得较多的差异蛋白质和生物学信息,证明了外泌体作为组学分析样本的价值和应用潜力。     

Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays

Proteomics is one of the most important issues in the post-genomic area, because it can greatly contribute to identifying protein biomarkers for disease diagnosis and drug screening. Protein array is a key technology for proteome researches and has been analyzed by various methods including fluorescence, mass spectrometry, atomic force microscopy and surface plasmon resonance (SPR). SPR biosensor is a promising technology in proteomics, since it has various advantages including real-time measurement of biomolecular interactions without labeling and the simple optical system for the device. SPR biosensors have a strong potential for analyzing proteomes by SPR imaging and SPR spectroscopic imaging, even though the challenge is to produce proteins on a proteomic scale.     

Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM

Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.     

Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.     

New method for prefractionation of plasma for proteomic analysis

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high‐abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high‐abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow? technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2‐DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.     

Determination of Fucose Concentration in a Lectin-Based Displacement Microfluidic Assay

We compare three different methods to quantify the monosaccharide fucose in solutions using the displacement of a large glycoprotein, lactoferrin. Two microfluidic analysis methods, namely fluorescence detection of (labeled) lactoferrin as it is displaced by unlabeled fucose and the displacement of (unlabeled) lactoferrin in SPR, provide fast responses and continuous data during the experiment, theoretically providing significant information regarding the interaction kinetics between the saccharide groups and binding sites. For comparison, we also performed a static displacement ELISA. The stationary binding site in all cases was immobilized S2-AAL, a monovalent polypeptide based on Aleuria aurantia lectin. Although all three assays showed a similar dynamic range, the microfluidic assays with fluorescent or SPR detection show an advantage in short analysis times. Furthermore, the microfluidic displacement assays provide a possibility to develop a one-step analytical platform.

     

Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins. Analysis of tryptic peptides was accomplished with microfluidic HPLC-Chip/MS system. Results indicate that high-abundant protein depletion combined with RP fractionation of plasma showed an improved dynamic range for proteomic analysis and enabled the identification of low-abundant plasma proteins.     

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography–triple quadrupole mass spectrometry‐based quantitative protein assays use bottom‐up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top‐down reversed‐phase liquid chromatography–triple quadrupole mass spectrometry method for analysis of cancer‐related intact proteins was evaluated. Seven growth factors (5.5–26.5 kDa; isoelectric points: 4.6–9.9) were analyzed on a wide‐pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1–500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.     

Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres

A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2 M NaCl, 100 mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100 ng mL⁻¹) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.