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ABSTRACT: Despite major advances in T cell receptor (TCR) biology and structure, how peptide-MHC complex (pMHC) ligands trigger αβ TCR activation remains unresolved. Two views exist. One model postulates that monomeric TCR-pMHC ligation events are sufficient while a second proposes that TCR-TCR dimerization in cis via Cα domain interaction plus pMHC binding is critical. We scrutinized 22 known TCR/pMHC complex crystal structures, and did not find any predicted molecular Cα-Cα contacts in these crystals that would allow for physiological TCR dimerization. Moreover, the presence of conserved glycan adducts on the outer face of the Cα domain preclude the hypothesized TCR dimerization through the Cα domain. Observed functional consequences of Cα mutations are likely indirect, with TCR microclusters at the immunological synapse driven by TCR transmembrane/cytoplasmic interactions via signaling molecules, scaffold proteins, and/or cytoskeletal elements.
Frontiers in immunology. 01/2013; 4:16.
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ABSTRACT: Self versus non-self discrimination is at the core of T-lymphocyte recognition. To this end, αβ T-cell receptors (TCRs) ligate 'foreign' peptides bound to major histocompatibility complex (MHC) class I or class II molecules (pMHC) arrayed on the surface of antigen-presenting cells (APCs). Since the discovery of TCRs approximately 30 years ago, considerable structural and functional data have detailed the molecular basis of their extraordinary ligand specificity and sensitivity in mediating adaptive T-cell immunity. This review focuses on the structural biology of the Fab-like TCRαβ clonotypic heterodimer and its unique features in conjunction with those of the associated CD3εγ and CD3εδ heterodimeric molecules, which, along with CD3ζζ homodimer, comprise the TCR complex in a stoichiometry of 1:1:1:1. The basis of optimized TCRαβ docking geometry on the pMHC linked to TCR mechanotransduction and required for T-cell signaling as well as CD4 and CD8 co-receptor function is detailed. A model of the TCR ectodomain complex including its connecting peptides suggests how force generated during T-cell immune surveillance and at the immunological synapse results in dynamic TCR quaternary change involving its heterodimeric components. Potential insights from the structural biology relevant to immunity and immunosuppression are revealed.
Immunological Reviews 11/2012; 250(1):102-19. · 11.15 Impact Factor
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ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons.
BMC Genomics 08/2012; 13(1):378. · 4.07 Impact Factor
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ABSTRACT: Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-γ was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-γ-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes.
Experimental Dermatology 08/2012; 21(8):625-9. · 3.54 Impact Factor
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ABSTRACT: We demonstrate a stochastic based method for performing active rheology using optical tweezers. By monitoring the displacement of an embedded particle in response to stochastic optical forces, a rapid estimate of the frequency dependent shear moduli of a sample is achieved in the range of 10(-1)-10(3) Hz. We utilize the method to probe linear viscoelastic properties of hydrogels at varied cross-linker concentrations. Combined with fluorescence imaging, our method demonstrates non-linear changes of bond strength between T cell receptors and an antigenic peptide due to force-induced cell activation.
Applied Physics Letters 07/2012; 101(3):31902. · 3.84 Impact Factor
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ABSTRACT: Mechanotransduction is a basis for receptor signaling in many biological systems. Recent data based upon optical tweezer experiments suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into biochemical signals upon specific peptide-MHC complex (pMHC) ligation. Tangential force applied along the pseudo-twofold symmetry axis of the TCR complex post-ligation results in the αβ heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell-APC interface. Accompanying TCR quaternary change likely fosters signaling via the lipid bilayer predicated on the magnitude and direction of the TCR-pMHC force. TCR glycans may modulate quaternary change, thereby altering signaling outcome as might the redox state of the CxxC motifs located proximal to the TM segments in the heterodimeric CD3 subunits. Predicted alterations in TCR TM segments and surrounding lipid will convert ectodomain ligation into the earliest intracellular signaling events.
Frontiers in immunology. 01/2012; 3:76.
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ABSTRACT: PB1-F2 is a major virulence factor of influenza A. This protein is a product of an alternative reading frame in the PB1-encoding RNA segment 2. Its presence of is dictated by the presence or absence of premature stop codons. This virulence factor is present in every influenza pandemic and major epidemic of the 20th century. Absence of PB1-F2 is associated with mild disease, such as the 2009 H1N1 ("swine flu").
The analysis of 8608 segment 2 sequences showed that only 8.5% have been annotated for the presence of PB1-F2. Our analysis indicates that 75% of segment 2 sequences are likely to encode PB1-F2. Two major populations of PB1-F2 are of lengths 90 and 57 while minor populations include lengths 52, 63, 79, 81, 87, and 101. Additional possible populations include the lengths of 59, 69, 81, 95, and 106. Previously described sequences include only lengths 57, 87, and 90. We observed substantial variation in PB1-F2 sequences where certain variants show up to 35% difference to well-defined reference sequences. Therefore this dataset indicates that there are many more variants that need to be functionally characterized.
Our web-accessible tool PB1-F2 Finder enables scanning of influenza sequences for potential PB1-F2 protein products. It provides an initial screen and annotation of PB1-F2 products. It is accessible at http://cvc.dfci.harvard.edu/pb1-f2.
BMC Bioinformatics 11/2011; 12 Suppl 13:S6. · 2.75 Impact Factor
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ABSTRACT: Functional cognate T cell recognition is mediated via the interaction of a T cell receptor complex with its pMHC ligand. Adams et al. (2011) in this issue of Immunity provide evidence that docking geometry may impact 2D binding and T cell activation.
Immunity 11/2011; 35(5):658-60. · 21.64 Impact Factor
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Mikyung Kim,
Zhen-Yu J Sun,
Kasper D Rand,
Xiaomeng Shi,
Likai Song,
Yuxing Cheng,
Amr F Fahmy,
Shreoshi Majumdar,
Gilad Ofek,
Yongping Yang,
Peter D Kwong,
Jia-Huai Wang,
John R Engen,
Gerhard Wagner, Ellis L Reinherz
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ABSTRACT: Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.
Nature Structural & Molecular Biology 11/2011; 18(11):1235-43. · 12.71 Impact Factor
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ABSTRACT: IκBNS has been identified as a member of the IκB family of NF-κB inhibitors, which undergoes induction upon TCR signaling. Mice carrying a targeted gene disruption of IκBNS demonstrate dysregulation of cytokines in T cells, macrophages, and dendritic cells. IκBNS mediates both positive and negative gene regulation, depending on individual cell type and/or cytokine. In this study, we demonstrate an additional role for IκBNS in the B cell lineage. B cells from IκBNS knockout (KO) mice were impaired in proliferative responses to LPS and anti-CD40. IgM and IgG3 Igs were drastically reduced in the serum of IκBNS KO mice, although IκBNS KO B cells exhibited a higher level of surface IgM than that found in wild-type mice. Switching to IgG3 was significantly reduced in IκBNS KO B cells. The in vitro induction of plasma cell development demonstrated that progression to Ab-secreting cells was impaired in IκBNS KO B cells. In agreement with this finding, the number of Ab-secreting cells in the spleens of IκBNS KO mice was reduced and production of Ag-specific Igs was lower in IκBNS KO mice after influenza infection as compared with wild-type mice. Additionally, IκBNS KO mice lacked B1 B cells and exhibited a reduction in marginal zone B cells. Thus, IκBNS significantly impacts the development and functions of B cells and plasma cells.
The Journal of Immunology 09/2011; 187(8):3942-52. · 5.79 Impact Factor
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ABSTRACT: The immune system is characterized by high combinatorial complexity that necessitates the use of specialized computational tools for analysis of immunological data. Machine learning (ML) algorithms are used in combination with classical experimentation for the selection of vaccine targets and in computational simulations that reduce the number of necessary experiments. The development of ML algorithms requires standardized data sets, consistent measurement methods, and uniform scales. To bridge the gap between the immunology community and the ML community, we designed a repository for machine learning in immunology named Dana-Farber Repository for Machine Learning in Immunology (DFRMLI). This repository provides standardized data sets of HLA-binding peptides with all binding affinities mapped onto a common scale. It also provides a list of experimentally validated naturally processed T cell epitopes derived from tumor or virus antigens. The DFRMLI data were preprocessed and ensure consistency, comparability, detailed descriptions, and statistically meaningful sample sizes for peptides that bind to various HLA molecules. The repository is accessible at http://bio.dfci.harvard.edu/DFRMLI/.
Journal of immunological methods 07/2011; 374(1-2):18-25. · 2.35 Impact Factor
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Karen S Anderson,
Wanyong Zeng,
Tetsuro Sasada,
Jaewon Choi,
Angelika B Riemer,
Mei Su,
Donna Drakoulakos,
Yoon-Joong Kang,
Vladimir Brusic,
Catherine Wu, Ellis L Reinherz
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ABSTRACT: Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1(26-34) (M26) and Cyp1B1(239-247) (Cyp239)] and one derived from the influenza A viral antigen [FluM1(58-66) (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.
Cancer Immunology and Immunotherapy 03/2011; 60(6):857-67. · 3.70 Impact Factor
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ABSTRACT: Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund's adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.
Biomaterials 02/2011; 32(14):3666-78. · 7.40 Impact Factor
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ABSTRACT: The αβ T cell receptor (TCR) is a multimeric complex whose β chain plays a crucial role in thymocyte development as well as antigen recognition by mature T lymphocytes. We report here crystal structures of individual β subunits, termed N15β (Vβ5.2Dβ2Jβ2.6Cβ2) and N30β (Vβ13Dβ1Jβ1.1Cβ2), derived from two αβ TCRs specific for the immunodominant vesicular stomatitis virus octapeptide (VSV-8) bound to the murine H-2K(b) MHC class I molecule. The crystal packing of the N15β structure reveals a homodimer formed through two Vβ domains. The Vβ/Vβ module is topologically very similar to the Vα/Vβ module in the N15αβ heterodimer. By contrast, in the N30β structure, the Vβ domain's external hydrophobic CFG face is covered by the neighboring molecule's Cβ domain. In conjunction with systematic investigation of previously published TCR single-subunit structures, we identified several conserved residues forming a concave hydrophobic patch at the center of the CFG outer face of the Vβ and other V-type Ig-like domains. This hydrophobic patch is shielded from solvent exposure in the crystal packing, implying that it is unlikely to be thermodynamically stable if exposed on the thymocyte surface. Accordingly, we propose a dimeric pre-TCR model distinct from those suggested previously by others and discuss its functional and structural implications.
Frontiers in immunology. 01/2011; 2:5.
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2011 IEEE International Conference on Bioinformatics and Biomedicine Workshops (BIBMW), Atlanta, GA, USA, November 12-15, 2011; 01/2011
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ABSTRACT: Persistent infection with high-risk human papilloma viruses (HPV) is the worldwide cause of many cancers, including cervical, anal, vulval, vaginal, penile, and oropharyngeal. Since T cells naturally eliminate the majority of chronic HPV infections by recognizing epitopes displayed on virally altered epithelium, we exploited Poisson detection mass spectrometry (MS(3)) to identify those epitopes and inform future T cell-based vaccine design. Nine cervical cancer biopsies from HPV-16 positive HLA-A(*)02 patients were obtained, histopathology determined, and E7 oncogene PCR-amplified from tumor DNA and sequenced. Conservation of E7 oncogene coding segments was found in all tumors. MS(3) analysis of HLA-A(*)02 immunoprecipitates detected E7(11-19) peptide (YMLDLQPET) in seven of the nine tumor biopsies. The remaining two samples were E7(11-19) negative and lacked the HLA-A(*)02 binding GILT thioreductase peptide despite possessing binding-competent HLA-A(*)02 alleles. Thus, the conserved E7(11-19) peptide is a dominant HLA-A(*)02 binding tumor antigen in HPV-16 transformed cervical squamous and adenocarcinomas. Findings that a minority of HLA-A(*)02:01 tumors lack expression of both E7(11-19) and a peptide from a thioreductase important in processing of cysteine-rich proteins like E7 underscore the value of physical detection, define a potential additional tumor escape mechanism and have implications for therapeutic cancer vaccine development.
Frontiers in immunology. 01/2011; 2:75.
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ABSTRACT: Broad coverage of the pathogen population is particularly important when designing CD8+ T-cell epitope vaccines against viral pathogens. Traditional approaches are based on combinations of highly conserved T-cell epitopes. Peptide block entropy analysis is a novel approach for assembling sets of broadly covering antigens. Since T-cell epitopes are recognized as peptides rather than individual residues, this method is based on calculating the information content of blocks of peptides from a multiple sequence alignment of homologous proteins rather than using the information content of individual residues. The block entropy analysis provides broad coverage of variant antigens. We applied the block entropy analysis method to the proteomes of the four serotypes of dengue virus (DENV) and found 1,551 blocks of 9-mer peptides, which cover 99% of available sequences with five or fewer unique peptides. In contrast, the benchmark study by Khan et al. (2008) resulted in 165 conserved 9-mer peptides. Many of the conserved blocks are located consecutively in the proteins. Connecting these blocks resulted in 78 conserved regions. Of the 1551 blocks of 9-mer peptides 110 comprised predicted HLA binder sets. In total, 457 subunit peptides that encompass the diversity of all sequenced DENV strains of which 333 are T-cell epitope candidates.
Frontiers in immunology. 01/2011; 2:69.
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ABSTRACT: The flavivirus genus is unusually large, comprising more than 70 species, of which more than half are known human pathogens. It includes a set of clinically relevant infectious agents such as dengue, West Nile, yellow fever, and Japanese encephalitis viruses. Although these pathogens have been studied exten-sively, safe and efficient vaccines lack for the majority of the flaviviruses. We have assembled a database that combines antigenic data of flaviviruses, specialized analysis tools, and workflows for automated complex analyses focusing on applications in immunology and vaccinology. FLAVIdB contains 12,858 entries of flavivirus antigen sequences, 184 verified T-cell epitopes, 201 verified B-cell epitopes, and 4 representative molecular structures of the dengue virus envelope protein. FLAVIdB was assembled by collection, annotation, and integration of data from GenBank, GenPept, UniProt, IEDB, and PDB. The data were subject to extensive quality control (redundancy elimination, error detection, and vocabulary consolidation). Further annotation of selected functionally relevant features was performed by organizing information extracted from the literature. The database was incorporated into a web-accessible data mining system, combining specialized data analysis tools for integrated analysis of relevant data categories (protein sequences, macromolecular structures, and immune epitopes). The data mining system includes tools for variability and conservation analysis, T-cell epitope prediction, and characterization of neutralizing components of B-cell epitopes. FLAVIdB is accessible at cvc.dfci.harvard.edu/flavi/ FLAVIdB represents a new generation of databases in which data and tools are integrated into a data min-ing infrastructures specifically designed to aid rational vaccine design by discovery of vaccine targets.
Immunome Research 01/2011; 7(3):1-9.
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ABSTRACT: MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes. NetMHCpan and NetMHCIIpan are used as prediction engines. The 13 HLA Class I supertypes are A1, A2, A3, A24, B7, B8, B27, B44, B58, B62, C1, and C4. The 13 HLA Class II DR supertypes are DR1, DR3, DR4, DR6, DR7, DR8, DR9, DR11, DR12, DR13, DR14, DR15, and DR16. In total, MULTIPRED2 enables prediction of peptide binding to 1077 variants representing 26 HLA supertypes. MULTIPRED2 has visualization modules for mapping promiscuous T-cell epitopes as well as those regions of high target concentration - referred to as T-cell epitope hotspots. Novel graphic representations are employed to display the predicted binding peptides and immunological hotspots in an intuitive manner and also to provide a global view of results as heat maps. Another function of MULTIPRED2, which has direct relevance to vaccine design, is the calculation of population coverage. Currently it calculates population coverage in five major groups in North America. MULTIPRED2 is an important tool to complement wet-lab experimental methods for identification of T-cell epitopes. It is available at http://cvc.dfci.harvard.edu/multipred2/.
Journal of immunological methods 12/2010; 374(1-2):53-61. · 2.35 Impact Factor
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ABSTRACT: A nanospray MS(3) method deployed on a quadrupole linear ion trap hybrid can detect targeted peptides with high dynamic range and high sensitivity from complex mixtures without separations. The method uses a recognition algorithm that is a modification of the relative (Kullback-Leibler, KL) entropy characterization of probabilistic distance to detect if reference MS(3) fragmentation patterns are components of acquired MS(3) spectra. The recognition reflects the probabilistic structure of physical MS measurements unlike the Euclidean or inner product metrics widely used for comparing spectra. It capably handles spectra with a significant chemical ion background in contrast to the Euclidean metric or the direct relative entropy. The full nanospray MS(3) method allows both the detection and quantitation of targets without the need to obtain isotopically labeled standards. By avoiding chromatographic separations and its associated surface losses, the detection can be applied to complex samples on a very limited material scale. The methodology is illustrated by applications to the medically important problem of detecting targeted major histocompatibility complex (MHC) I associated peptides extracted from limited cell numbers.
Analytical Chemistry 10/2010; · 5.86 Impact Factor
