Surface plasmon resonance mass spectrometry in proteomics

Due to the enormous complexity of the proteome, focus in proteomics shifts more and more from the study of the complete proteome to the targeted analysis of part of the proteome. The isolation of this specific part of the proteome generally includes an affinity-based enrichment. Surface plasmon resonance (SPR), a label-free technique able to follow enrichment in real-time and in a semiquantitative manner, is an emerging tool for targeted affinity enrichment. Furthermore, in combination with mass spectrometry (MS), SPR can be used to both selectively enrich for and identify proteins from a complex sample. Here we illustrate the use of SPR-MS to solve proteomics-based research questions, describing applications that use very different types of immobilized components: such as small (drug or messenger) molecules, peptides, DNA and proteins. We evaluate the current possibilities and limitations and discuss the future developments of the SPR-MS technique.     

Surface plasmon resonance mass spectrometry: recent progress and outlooks

The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) has created a unique approach to protein investigations. Surface plasmon resonance is used to quantify interactions between proteins and surface-immobilized ligands, and MS is used to determine the structural features of the bound proteins. Recent progress in SPR-MS includes improved methods and operations, increased limits of detection, multi-protein analysis and protein-complex delineation. With the subsequent design of SPR protein arrays, SPR-MS is expected to enter into the field of high-throughput protein interaction discovery and miniaturized diagnostics.     

Combinations of SPR and MS for characterization of native and recombinant proteins in cell lysates

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni²⁺ for SPR analysis of the specific Ni²⁺ binding response and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein and of cauliflower were investigated for specific binding to the phosphopeptide ligands immobilized on a sensorchip by SPR. Subsequently, the bound protein was eluted and analyzed by MS for characterization of intact mass and peptide mass mapping.     

蛋白质组学技术及其在肺脏疾病研究中的应用

蛋白质组学是旨在研究蛋白质表达谱和蛋白质与蛋白质之间相互作用的新领域,其研究必须依赖高通量、高自动化的技术.简要介绍了蛋白质组分离技术(双向电泳、色谱),蛋白质组分析技术(质谱分析、氨基酸组成分析、蛋白质芯片,Edman降解法测N端序列),蛋白质相互作用技术(酵母双杂交系统、表面等离子共振)以及生物信息学.并从寻找差异表达的蛋白质,寻找用于诊断的疾病相关的标记分子,研究疾病的发病机制三方面介绍了蛋白质组技术在肺脏疾病研究中的应用.     

SPR-MS in functional proteomics

The mapping of protein networks and the establishment of thefunctional relationships between expressed proteins and theireffects on cellular processes represents a great challenge forfunctional or interaction proteomics. The combination of surfaceplasmon resonance (SPR)-based technology with mass spectrometry(MS) has created a unique analytical tool for functional proteomicsinvestigations. Proteins are affinity purified, quantified andcharacterised in terms of their interactions, while the massspectrometer identifies and structurally characterises the biomolecules.Recent developments have led to a closer integration of thesekey technologies, providing a combined approach which enablesidentification of proteins selected on the basis of their functionalbinding criteria. In addition to a historical overview of thisfield, some recent detailed examples of combined SPR-MS approacheswill be reviewed in a number of key application areas, includingligand fishing, peptide sequence and post-translational modificationanalysis by SPR-MS/MS and enzyme inhibitor screening.      

Characterization of the Mycobacterium tuberculosis proteome by liquid chromatography mass spectrometry-based proteomics techniques: a comprehensive resource for tuberculosis research

Approximately, one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Secreted and membrane proteins that interact with the host play important roles for the pathogenicity of the bacteria and are potential drug targets or components of vaccines. In this present study, subcellular fractionation in combination with membrane enrichment was used to comprehensively analyze the M. tuberculosis proteome. The proteome of the M. tuberculosis cell wall, membrane, cytosol, lysate, and culture filtrate was defined with a high coverage. Exceptional enrichment for membrane proteins was achieved using wheat germ agglutinin (WGA)-affinity two-phase partitioning, a technique that has to date not yet been exploited for the enrichment of mycobacterial membranes. Overall, 1051 M. tuberculosis protein groups including 183 transmembrane proteins have been identified by LC-MS/MS analysis using stringent database search criteria with a minimum of two peptides and an estimated FDR of less than 1%. With many mycobacterial antigens and lipoglycoproteins identified, the results from this study suggest that many of the newly discovered proteins could represent potential candidates mediating host-pathogen interactions. In addition, this data set provides experimental information about protein localization and thus serves as a valuable resource for M. tuberculosis proteome research.     

Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins

Mitochondria consist of four compartments-outer membrane, intermembrane space, inner membrane, and matrix--with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of approximately 85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sj?gren-Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments.     

Subcellular fractionation enhances proteome coverage of pancreatic duct cells

ObjectivesSubcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage.MethodsWe used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared – via mass spectrometry-based analysis – the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates.ResultsWe identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle.ConclusionsSubcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.     

Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.     

Head-to-head comparison of serum fractionation techniques

Multiple approaches for simplifying the serum proteome have been described. These techniques are generally developed across different laboratories, samples, mass spectrometry platforms, and analysis tools. Hence, comparing the available schemes is impossible from the existing literature because of confounding variables. We describe a head-to-head comparison of several serum fractionation schemes, including N-linked glycopeptide enrichment, cysteinyl-peptide enrichment, magnetic bead separation (C3, C8, and WCX), size fractionation, protein A/G depletion, and immunoaffinity column depletion of abundant serum proteins. Each technique was compared to results obtained from unfractionated human serum. The results show immunoaffinity subtraction is the most effective means for simplifying the serum proteome while maintaining reasonable sample throughput. The reported dataset is publicly available and provides a standard against which emergent technologies can be compared and evaluated for their contribution to serum-based biomarker discovery.     

Global survey of the bovine salivary proteome: integrating multidimensional prefractionation, targeted, and glycocapture strategies

Saliva is easily obtainable from a large number of animals in a noninvasive manner and contains a wide diversity of compounds including hormones, metabolites, and proteins that may be a good source of biomarkers of health and disease. Here we have used a combination of multidimensional prefractionation, targeted, and glycocapture methodologies to profile the bovine salivary proteome. The nontargeted approach used four different separation methodologies consisting of SDS-PAGE, Off-gel fractionation, RP-HPLC, and SCX-HPLC. In the targeted approach, we've employed a hypothesis-based methodology by only selecting extracellular proteins from in silico data. Finally, the hydrazide capture methodology not only enabled us to identify formerly N-linked glycoproteins but it also provided a selective enrichment process for the identification of low abundance proteins. Together, the three different approaches identified 402 salivary proteins and 45 N-linked glycoproteins. A large number of these proteins have previously been uncharacterized in bovine saliva. To date, this is the largest global survey of the bovine salivary proteome and expands the potential of the diagnostic utility of this fluid to guide development of experiments seeking biomarkers for health traits (i.e., disease resistance) as well as feed conversion efficiency and productivity traits in dairy and beef cattle.     

Characterization of the mouse brain proteome using global proteomic analysis complemented with cysteinyl-peptide enrichment

We report a global proteomic approach for analyzing brain tissue and for the first time a comprehensive characterization of the whole mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 nonredundant proteins ( approximately 34% of the predicted mouse proteome). A total of 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models. The proteomic approach presented here may have broad applications for rapid proteomic analyses of various mouse models of human brain diseases.     

Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry

Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of "fluorous proteomics," whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.     

Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column

The selective removal of high-abundance proteins is considered to be an important prerequisite for a sensitive proteome analysis in plasma. In this study, we examined the "multiaffinity removal system", an immunoaffinity depletion column targeted against six plasma proteins. As determined by sandwich ELISA, the depletion rate for each target protein is >99% over 200 cycles of regeneration. Our data give evidence that two column antibodies are slowly inactivated during the repeated use of the column; however, the individual depletion rate meets the specification of the manufacturer. To estimate a potential loss of analytes after the immunodepletion, we performed spiking/recovery experiments with a selection of tumor markers at concentrations in the lower to medium ng/mL range. The average recovery of 9 out of 11 markers is 78%. A significant proportion of two other markers binds to the column. Based on the average marker recovery and a depletion of ;85% of the total protein we estimate a five-fold enrichment of a potential biomarker by the use of this depletion column. We conclude that the selective depletion of plasma proteins by immunoaffinity chromatography is a valid strategy for the enrichment of potential biomarkers sought by proteomics methodologies.     

The cell wall subproteome of Listeria monocytogenes

The surface subproteome of Listeria monocytogenes that includes many proteins already known to be involved in virulence and interaction with host cells has been characterized. A new method for the isolation of a defined surface proteome of low complexity has been established based on serial extraction of proteins by different salts at high concentration, and in all 55 proteins were identified by N-terminal sequencing and mass spectrometry. About 16% of these proteins are of unknown function and three proteins have no orthologue in the nonpathogenic L. innocua and might be involved in virulence mechanisms. Remarkably, a relatively high number of proteins with a function in the cytoplasmic compartment was identified in this surface proteome. These proteins had neither predicted or detectable signal peptides nor could any modification be observed except removal of the N-terminal methionine. Enolase (Lmo2455) is one of these proteins. It was shown to be present in the cell wall of the pathogen by immunoelectron microscopy and, along with heat shock factor DnaK (Lmo1473), elongation factor TU (Lmo2653), and glyceraldehyde-3-phosphate dehydrogenase (Lmo2459), it was found to be able to bind human plasminogen in overlay blots and surface plasmon resonance (SPR) experiments. The KD values of these interactions were determined by SPR measurements. The data indicate a possible role of these proteins as receptors for human plasminogen on the bacterial cell surface. The potential role of this recruitment of a host protease for extracellular invasion mechanisms is discussed.     

Mapping yeast N-glycosites with isotopically recoded glycans

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.     

Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways

Introduction: Biomarkers are commonly used to stratify cancer patients and guide targeted therapies, but most biomarkers are of a genomic nature. Discrepancies between the genome and proteome and the high rates of drug resistance indicate that proteomic analyses may provide additional critically important information. Here we present immuno-Matrix-Assisted Laser Desorption/Ionization (iMALDI), the combination of immuno-affinity enrichment of peptides followed by direct MALDI-mass spectrometry analysis. iMALDI is a highly sensitive, targeted protein-quantitation technique with the potential to measure clinically relevant signaling-pathway proteins using minimal sample amounts, thus improving upon existing methodologies.

Areas covered: We provide a brief overview of the current state of biomarker analysis technologies for modern cancer treatment. We also show the advantages of iMALDI for translating potential new biomarkers into the clinic, factors to consider for iMALDI assay development, and the utility of iMALDI for the quantitation of cell-signaling proteins.

Expert commentary: We see targeted mass spectrometry approaches such as iMALDI as an important part of improving patient responses to targeted therapies by providing highly sensitive, accurate, precise, and specific measurements of signaling-pathway proteins, both in tumor cells and in cells from the tumor microenvironment. iMALDI results can be integrated with other -omics data to aid in tumor-targeting therapies and immuno-oncology.     

Analysis of the urine proteome via a combination of multi-dimensional approaches

Urine is a biological fluid that is non-invasively and easily harvested, and exhibits high stability from the proteomics point of view. At the downside, the overall low protein content of urine as well as the presence of low- and high-abundance proteins underscores the need for protein enrichment. As a continuation of previous efforts towards the comprehensive characterization of the urine proteome, the current study targeted the mining of urine proteins through the combined application of different protein separation methodologies, specifically, liquid chromatography and preparative electrophoresis along with 1D gel electrophoresis and protein identification by mass spectrometry. In order to enhance comparison and integration of different experimental data sets, the "standard" urine sample developed within the European Kidney and Urine Proteomics (EuroKUP) COST Action, was employed. As a contribution to the existing knowledge, we focused on maintaining and providing information about experimental mass of the identified proteins as well as information pertaining to their relative abundance--as allowed by technical limitations--thus providing an initial view of different isoforms representation and facilitating their future characterization. The difficulties in comparing proteome mining data sets become once more evident, underscoring the need for adopting standardized ways for data reporting as well as for potential new approaches for data analysis involving a thorough investigation of received information at the peptide level.