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In JoVE (1)
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- Molecular and Biochemical Parasitology
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- PLoS Neglected Tropical Diseases
- Molecular & Cellular Proteomics : MCP
- PLoS Pathogens
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Articles by Terry W. Pearson in JoVE
JoVE General
Kvantifiering av proteiner med Peptide Immunoaffinity Anrikning kombination med masspektrometri
1Clinical Research Division, Fred Hutchinson Cancer Research Center - FHCRC, 2Department of Biochemistry and Microbiology, University of Victoria, 3Broad Institute of MIT and Harvard, 4Genome BC Proteomics Centre, University of Victoria, 5Plasma Proteome Institute
Stabila isotopen standarder och Capture av Anti-peptidantikroppar (SISCAPA) par affinitet anrikning av peptider med stabila isotoputspädning masspektrometri (MRM-MS) för att ge kvantitativ mätning av peptider som substitut för sina respektive proteiner. Här beskriver vi det protokoll som använder magnetiska partiklar i en delvis automatiserad format.
Other articles by Terry W. Pearson on PubMed
Glycosylphosphatidylinositol-anchored Surface Molecules of Trypanosoma Congolense Insect Forms Are Developmentally Regulated in the Tsetse Fly
Procyclic culture forms of Trypanosoma congolense have been shown to express a glutamic acid/alanine-rich protein (GARP) on their surface. By labelling T. congolense procyclic culture forms with glycosylphosphatidylinositol (GPI) precursors, we show that GARP is bound to the membrane by a GPI anchor and demonstrate the presence of two additional GPI-anchored surface molecules of 24-34 and 58 kDa that are abundantly expressed. The 24-34 kDa molecule, which is recognised by monoclonal antibodies that bind to the surface of living trypanosomes, is resistant to proteolysis, suggesting that it consists (predominantly) of non-proteinaceous material. We have therefore named it protease-resistant surface molecule (PRS). In common with the EP and GPEET procyclins of Trypanosoma brucei, the relative expression of the T. congolense GPI-anchored molecules changes during parasite development in the tsetse fly. PRS is abundantly expressed by procyclic trypanosomes in the midgut shortly after infection, but is downregulated in established midgut forms and completely absent from the epimastigote form in the proboscis. In contrast, GARP is downregulated in parasites in the tsetse fly midgut, but upregulated in the epimastigote form. Unexpectedly, 14 days post-infection, procyclic forms frequently are negative for both PRS and GARP, suggesting that they might be expressing another stage-specific surface antigen at this point in the life cycle.
The African Trypanosome Cyclophilin A Homologue Contains Unusual Conserved Central and N-terminal Domains and is Developmentally Regulated
We have cloned and characterized the homologue of cyclophilin A (CypA) from Trypanosoma brucei brucei, Trypanosoma congolense, Trypanosoma evansi and Trypanosoma vivax. The 1-kilobase African trypanosome CypA complementary DNA contains an open reading frame of 531 base pairs, corresponding to 177 amino acids with a calculated molecular weight of 18,700. The CypA gene is present at one copy/haploid genome in T. brucei, T. congolense and T. vivax and is located on large chromosomes (>3 Mb) in T. brucei. CypA is differentially transcribed in African trypanosomes and is localized in the cytosol as well as in the flagellum. It is also detected in the supernatant of in vitro cultivated parasites. The African trypanosome CypA is unique due to a ten amino acid residue N-terminus extension and a block that includes a three amino acid insertion around position 100 that might result in a differently structured surface. Wild-type recombinant CypA and several mutants were over-expressed in Escherichia coli and purified to >98% homogeneity. Antisera from cattle immunized with a trypanosome fraction containing immunosuppressive activity react strongly against CypA. These data indicate that trypanosome CypA might play an important role in the establishment and maintenance of infections in susceptible animals.
Cationic Antimicrobial Peptide Killing of African Trypanosomes and Sodalis Glossinidius, a Bacterial Symbiont of the Insect Vector of Sleeping Sickness
Nine biochemically distinct cationic antimicrobial peptides were tested in vitro for their effects on bloodstream forms and procyclic (insect) forms of African trypanosomes, the protozoan parasites that cause African sleeping sickness in humans and trypanosomiasis in domestic animals. At low concentrations, one peptide completely inhibited growth of bloodstream forms, one inhibited procyclic forms, and five inhibited both trypanosome life cycle stages. The peptides were also tested on Sodalis glossinidius, a bacterial symbiont of tsetse flies. S. glossinidius was highly resistant to seven of the nine peptides, including both that specifically inhibited either bloodstream or procyclic forms and three of the five that inhibited both trypanosome life cycle stages. The results indicate that several of these peptides may be ideal candidates for therapy of trypanosome infected mammals or for transgenic expression in S. glossinidius as a strategy for inhibiting trypanosome survival, development, and maturation in tsetse and interference with transmission of African sleeping sickness.
An Effective and Rapid Method for Functional Characterization of Immunoadsorbents Using POROS Beads and Flow Cytometry
To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.
Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA)
A method (denoted SISCAPA) for quantitation of peptides in complex digests is described. In the method, anti-peptide antibodies immobilized on 100 nanoliter nanoaffinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled). In a series of pilot experiments, tryptic test peptides were chosen for four proteins of human plasma (hemopexin, alpha1 antichymotrypsin, interleukin-6, and tumor necrosis factor-alpha) from a pool of 10,203 in silico tryptic peptide candidates representing 237 known plasma components. Rabbit polyclonal antibodies raised against the chosen peptide sequences were affinity purified and covalently immobilized on POROS supports. Binding and elution from these supports was shown to provide an average 120-fold enrichment of the antigen peptide relative to others, as measured by selected ion monitoring (SIM) or selected reaction monitoring (SRM) electrospray mass spectrometry. The columns could be recycled with little loss in binding capacity, and generated peptide ion current measurements with cycle-to-cycle coefficients of variation near 5%. Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays. The method appears suitable for rapid generation of assays for defined proteins, and should find application in the validation of diagnostic protein panels in large sample sets.
Identification of a Functioning Mitochondrial Uncoupling Protein 1 in Thymus
We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent B(max) and K(D) values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus.
Inhibition of PACAP Activity by a Receptor Antagonist Results in Changes in Cell Cycle and Apoptotic Proteins in Chick Neuroblasts
We showed previously that early chick neuroblasts stop proliferating and undergo apoptosis when deprived of endogenous pituitary adenylate cyclase-activating polypeptide (PACAP). To identify proteins involved in these processes, we blocked the primary PACAP receptor and determined protein changes using isotope-coded affinity tag (ICAT) analysis. Cell cycle exit was characterized by a decrease in proteins regulating ribosome biogenesis and protein translation. Apoptosis was linked directly to a tumor suppressor that increases apoptosome activity and indirectly to reduced mitochondrial activity. ICAT analysis, combined with flow cytometric analysis, suggested that some cells were differentiating, rather than undergoing apoptosis. In summary, we have confirmed that withdrawal of PACAP from early chick neuroblasts causes cell cycle exit and apoptosis, and identified proteins involved in proliferation, exit, apoptosis, and possibly differentiation.
On the Mechanism of Mitochondrial Uncoupling Protein 1 Function
Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.
Tumour Immunity and T Cell Memory Are Induced by Low Dose Inoculation with a Non-replicating Adenovirus Encoding TAP1
Despite continued progress in understanding the pathophysiology of tumours, curative therapeutic options are still lacking for the metastatic form of the disease. One approach that has gathered considerable interest is the creation of therapeutic vaccines using genetically engineered non-replicating viruses as vehicles to revive immunosurveillance mechanisms that may eradicate residual tumour cells. A perceived problem with this approach is that the number of non-replicating viruses used as a vaccine inoculum does not remotely approximate the total number of cells in the body, nor even the number of tumour cells in the case of large tumour burden or metastasis. Here, we addressed the hypothesis that a limited amount of inoculum (1x10(8) PFU) of recombinant non-replicating adenovirus encoding human TAP1 (AdhTAP1) can induce protective immunity against 1.5x10(5) TAP-deficient, metastatic melanoma cells transplanted into a normal mouse (total of approximately 1x10(11) body cells). We show that efficacious anti-tumour cytolytic T cell responses are indeed induced by injecting melanoma-bearing animals with small numbers of recombinant viruses, resulting in increases in tumour-infiltrating dendritic cells, enhanced memory T cell subpopulations and, most importantly, in increased animal survival. This novel approach uses a limited input inoculum relative to the tumour cell mass, and thus achieves an efficacious outcome that has so far eluded other vaccine, immunotherapeutic or gene therapeutic strategies where there is a requisite for the majority of tumour cells to be transduced for beneficial outcome to be achieved.
Infections with Immunogenic Trypanosomes Reduce Tsetse Reproductive Fitness: Potential Impact of Different Parasite Strains on Vector Population Structure
The parasite Trypanosoma brucei rhodesiense and its insect vector Glossina morsitans morsitans were used to evaluate the effect of parasite clearance (resistance) as well as the cost of midgut infections on tsetse host fitness. Tsetse flies are viviparous and have a low reproductive capacity, giving birth to only 6-8 progeny during their lifetime. Thus, small perturbations to their reproductive fitness can have a major impact on population densities. We measured the fecundity (number of larval progeny deposited) and mortality in parasite-resistant tsetse females and untreated controls and found no differences. There was, however, a typanosome-specific impact on midgut infections. Infections with an immunogenic parasite line that resulted in prolonged activation of the tsetse immune system delayed intrauterine larval development resulting in the production of fewer progeny over the fly's lifetime. In contrast, parasitism with a second line that failed to activate the immune system did not impose a fecundity cost. Coinfections favored the establishment of the immunogenic parasites in the midgut. We show that a decrease in the synthesis of Glossina Milk gland protein (GmmMgp), a major female accessory gland protein associated with larvagenesis, likely contributed to the reproductive lag observed in infected flies. Mathematical analysis of our empirical results indicated that infection with the immunogenic trypanosomes reduced tsetse fecundity by 30% relative to infections with the non-immunogenic strain. We estimate that a moderate infection prevalence of about 26% with immunogenic parasites has the potential to reduce tsetse populations. Potential repercussions for vector population growth, parasite-host coevolution, and disease prevalence are discussed.
Anti-peptide Antibody Screening: Selection of High Affinity Monoclonal Reagents by a Refined Surface Plasmon Resonance Technique
A refined surface plasmon resonance method was developed to measure the kinetics of peptide binding to rabbit monoclonal antibodies (RabMAbs). Optimized amounts of RabMAbs were captured onto sensor chips from hybridoma supernatants followed by binding of free peptides from solution. This allowed kinetic measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies and determination of affinity constants without complications contributed by avidity considerations. Peptide-binding responses were normalized for the amount of antibody present in each sample and a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants ka and kd, and the affinity constant KD (kd/ka), could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of peptide antigens were performed to validate the reliability of single-concentration measurements. By combining data on affinity, activity and concentration, ranking of the antibody-containing supernatants was performed, allowing selection of high quality RabMAbs for binding of peptides in solution.
A Human Proteome Detection and Quantitation Project
The lack of sensitive, specific, multiplexable assays for most human proteins is the major technical barrier impeding development of candidate biomarkers into clinically useful tests. Recent progress in mass spectrometry-based assays for proteotypic peptides, particularly those with specific affinity peptide enrichment, offers a systematic and economical path to comprehensive quantitative coverage of the human proteome. A complete suite of assays, e.g. two peptides from the protein product of each of the approximately 20,500 human genes (here termed the human Proteome Detection and Quantitation project), would enable rapid and systematic verification of candidate biomarkers and lay a quantitative foundation for subsequent efforts to define the larger universe of splice variants, post-translational modifications, protein-protein interactions, and tissue localization.
Killing of Trypanosomatid Parasites by a Modified Bovine Host Defense Peptide, BMAP-18
Tropical diseases caused by parasites continue to cause socioeconomic devastation that reverberates worldwide. There is a growing need for new control measures for many of these diseases due to increasing drug resistance exhibited by the parasites and problems with drug toxicity. One new approach is to apply host defense peptides (HDP; formerly called antimicrobial peptides) to disease control, either to treat infected hosts, or to prevent disease transmission by interfering with parasites in their insect vectors. A potent anti-parasite effector is bovine myeloid antimicrobial peptide-27 (BMAP-27), a member of the cathelicidin family. Although BMAP-27 is a potent inhibitor of microbial growth, at higher concentrations it also exhibits cytotoxicity to mammalian cells. We tested the anti-parasite activity of BMAP-18, a truncated peptide that lacks the hydrophobic C-terminal sequence of the BMAP-27 parent molecule, an alteration that confers reduced toxicity to mammalian cells.
SISCAPA Peptide Enrichment on Magnetic Beads Using an In-line Bead Trap Device
A SISCAPA (stable isotope standards and capture by anti-peptide antibodies) method for specific antibody-based capture of individual tryptic peptides from a digest of whole human plasma was developed using a simplified magnetic bead protocol and a novel rotary magnetic bead trap device. Following off-line equilibrium binding of peptides by antibodies and subsequent capture of the antibodies on magnetic beads, the bead trap permitted washing of the beads and elution of bound peptides inside a 150-microm-inner diameter capillary that forms part of a nanoflow LC-MS/MS system. The bead trap sweeps beads against the direction of liquid flow using a continuous succession of moving high magnetic field-gradient trap regions while mixing the beads with the flowing liquid. This approach prevents loss of low abundance captured peptides and allows automated processing of a series of SISCAPA reactions. Selected tryptic peptides of alpha(1)-antichymotrypsin and lipopolysaccharide-binding protein were enriched relative to a high abundance serum albumin peptide by 1,800 and 18,000-fold, respectively, as measured by multiple reaction monitoring. A large majority of the peptides that are bound nonspecifically in SISCAPA reactions were shown to bind to components other than the antibody (e.g. the magnetic beads), suggesting that substantial improvement in enrichment could be achieved by development of improved inert bead surfaces.
Tsetse EP Protein Protects the Fly Midgut from Trypanosome Establishment
African trypanosomes undergo a complex developmental process in their tsetse fly vector before transmission back to a vertebrate host. Typically, 90% of fly infections fail, most during initial establishment of the parasite in the fly midgut. The specific mechanism(s) underpinning this failure are unknown. We have previously shown that a Glossina-specific, immunoresponsive molecule, tsetse EP protein, is up regulated by the fly in response to gram-negative microbial challenge. Here we show by knockdown using RNA interference that this tsetse EP protein acts as a powerful antagonist of establishment in the fly midgut for both Trypanosoma brucei brucei and T. congolense. We demonstrate that this phenomenon exists in two species of tsetse, Glossina morsitans morsitans and G. palpalis palpalis, suggesting tsetse EP protein may be a major determinant of vector competence in all Glossina species. Tsetse EP protein levels also decline in response to starvation of the fly, providing a possible explanation for increased susceptibility of starved flies to trypanosome infection. As starvation is a common field event, this fact may be of considerable importance in the epidemiology of African trypanosomiasis.
Mass-spectrometry-based Clinical Proteomics--a Review and Prospective
This review reports on the current and emerging technologies for the use of mass-spectrometry-based proteomics in clinical applications.
MALDI Immunoscreening (MiSCREEN): a Method for Selection of Anti-peptide Monoclonal Antibodies for Use in Immunoproteomics
A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.
Evaluation of Large Scale Quantitative Proteomic Assay Development Using Peptide Affinity-based Mass Spectrometry
Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/μl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.
Differential Protein Expression Throughout the Life Cycle of Trypanosoma Congolense, a Major Parasite of Cattle in Africa
Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis. Three complete biological replicates of all four life cycle stages were produced from T. congolense IL3000, a cloned parasite that is amenable to culture of major life cycle stages in vitro. Cellular proteins from each life cycle stage were trypsin digested and the resulting peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ). The peptides were then analyzed by tandem mass spectrometry (MS/MS). This method was used to identify and relatively quantify proteins from the different life cycle stages in the same experiment. A search of the Wellcome Trust's Sanger Institute's semi-annotated T. congolense database was performed using the MS/MS fragmentation data to identify the corresponding source proteins. A total of 2088 unique protein sequences were identified, representing 23% of the ∼9000 proteins predicted for the T. congolense proteome. The 1291 most confidently identified proteins were prioritized for further study. Of these, 784 yielded annotated hits while 501 were described as "hypothetical proteins". Six proteins showed no significant sequence similarity to any known proteins (from any species) and thus represent new, previously uncharacterized T. congolense proteins. Of particular interest among the remainder are several membrane molecules that showed drastic differential expression, including, not surprisingly, the well-studied variant surface glycoproteins (VSGs), invariant surface glycoproteins (ISGs) 65 and 75, congolense epimastigote specific protein (CESP), the surface protease GP63, an amino acid transporter, a pteridine transporter and a haptoglobin-hemoglobin receptor. Several of these surface disposed proteins are of functional interest as they are necessary for survival of the parasites.
Structural Characterization and Epitope Mapping of the Glutamic Acid/alanine-rich Protein from Trypanosoma Congolense: Defining Assembly on the Parasite Cell Surface
Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.
Inter-laboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma
The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to MRM-MS produces immuno-MRM assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the inter-laboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay CV, LOD, LOQ and recovery was evaluated. Limits of detection were at or below 1 ng/mL for the assayed proteins in 30 uL of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and inter-laboratory CVs above the LOQ of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope labeled protein as an internal standard instead of stable isotope labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.
Precision of Heavy-Light Peptide Ratios Measured by MALDI-TOF Mass Spectrometry
We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.
