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  • 1
    Abstract: Proteomic approaches are of growing importance in the biologist's toolbox. It greatly benefited from past and recent advances in sampling, chemical processing, mass spectrometry (MS) instrumentation, and data processing. MS-based analysis of proteins is now in the process of being translated in pathology for objective diagnoses. In this viewpoint, we present the workflows that we think are the most promising for applications in pathology. We also comment what we think are prerequisites for a successful translational implementation.
    Type of Publication: Journal article published
    PubMed ID: 29236356
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  • 2
    Keywords: PEPTIDE ; SPECTRA ; Germany ; PROTEIN ; PROTEINS ; data mining ; PHOSPHORYLATION ; SEQUENCE ; VARIANTS ; ACID ; CLEAVAGE ; IDENTIFICATION ; ELECTROSPRAY ; mass spectrometry ; tandem mass spectrometry ; fragmentation ; DATABASE ; MASS-SPECTROMETRY ; PEPTIDES ; STRATEGIES ; MASSES ; HIGH-RESOLUTION ; EXTRACTION ; intensity ; PHOSPHORYLATION SITES ; covalent modification ; N-terminal acetylation ; POSITIVE-ION MODE ; sequence isoforms
    Abstract: Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a(2), b(2), b(3), y(1), y(2), and y(3). The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms
    Type of Publication: Journal article published
    PubMed ID: 15714472
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  • 3
    Keywords: CELLS ; Germany ; HYBRIDIZATION ; microarray ; ANTIGEN ; SIMULATION ; BINDING ; antibodies ; TRANSPORT ; ASSAY ; microarrays ; KINETICS ; sensitivity ; IMMUNOASSAYS ; LINKED-IMMUNOSORBENT-ASSAY ; PROTEIN MICROARRAYS ; DIFFUSION ; antibody microarray ; ASSAYS ; ANTIBODY MICROARRAYS ; SOLID SUPPORTS ; adsorption ; two-compartment model ; mass transport ; BIOSENSORS ; mass-transport limited reaction ; microspot kinetics
    Abstract: It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology
    Type of Publication: Journal article published
    PubMed ID: 16385475
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  • 4
    Keywords: CELLS ; CELL ; Germany ; PATHWAYS ; QUANTIFICATION ; SYSTEM ; SYSTEMS ; microarray ; PROTEIN ; PROTEINS ; SAMPLES ; DRUG ; BIOLOGY ; SIGNAL ; ASSAY ; microarrays ; EFFICIENT ; ELECTROPHORESIS ; BIOPSY ; PROTEOMICS ; signaling ; RECOMBINANT ; RE ; CAPACITY ; ARRAY ; GELS ; analysis ; methods ; BIOPSIES ; signaling networks ; protein arrays ; protein quantification ; reverse phase protein microarray
    Abstract: The advancement of efficient technologies to comply with the needs of systems biology and drug discovery has so far not received adequate attention. A substantial bottleneck for the time-resolved quantitative description of signaling networks is the limited throughput and the inadequate sensitivity of currently established methods. Here, we present an improved protein microarray-based approach towards the sensitive detection of proteins in the fg-range which is based on signal detection in the near-infrared range. The high sensitivity of the assay permits the specific quantification of proteins derived from as little as only 20 000 cells with an error rate of only 5%. The capacity is limited to the analysis of up to 500 different samples per microarray. Protein abundance is determined qualitatively, and quantitatively, if recombinant protein is available. This novel approach was called IPAQ (infrared-based protein arrays with quantitative readout). IPAQ offers a highly sensitive experimental approach superior to the established standard protein quantification technologies, and is suitable for quantitative proteomics. Employing the IPAQ approach, a detailed analysis of activated signaling networks in biopsy samples and of crosstalk between signaling modules as required in drug discovery strategies can easily be performed
    Type of Publication: Journal article published
    PubMed ID: 17309101
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  • 5
    Keywords: CELL ; Germany ; INFORMATION ; TOOL ; PROTEIN ; BIOLOGY ; SEQUENCE ; COLLISION-INDUCED DISSOCIATION ; TANDEM MASS-SPECTROMETRY ; PEPTIDES ; PROTEOMICS ; PROTEIN IDENTIFICATION ; PERFORMANCE LIQUID-CHROMATOGRAPHY ; HIGH-RESOLUTION ; AMINO-ACID-RESIDUES ; FRAGMENT ; CID ; BENEFIT ; Sequence information ; b ion series ; ELECTRON-TRANSFER DISSOCIATION ; IN-SOURCE DECAY ; LASER-DESORPTION IONIZATION ; Manual sequencing ; REVERSED-PHASE HPLC ; y ion series
    Abstract: The current status of de novo sequencing of peptides by MS/MS is reviewed with focus on collision cell MS/MS spectra. The relation between peptide structure and observed fragment ion series is discussed and the exhaustive extraction of sequence information from CID spectra of protonated peptide ions is described. The partial redundancy of the extracted sequence information and a high mass accuracy are recognized as key parameters for dependable de novo sequencing by MS. in addition, the benefits of special techniques enhancing the generation of long uninterrupted fragment ion series for de novo peptide sequencing are highlighted. Among these are terminal O-18 labeling, MSn of sodiated peptide ions, N-terminal derivatization, the use of special proteases, and time-delayed fragmentation. The emerging electron transfer dissociation technique and the recent progress of MALDI techniques for intact protein sequencing are covered. Finally, the integration of bioinformatic tools into peptide de novo sequencing is demonstrated
    Type of Publication: Journal article published
    PubMed ID: 19953542
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  • 6
    Keywords: EXPRESSION ; Germany ; QUANTIFICATION ; PROTEIN ; PROTEINS ; BIOLOGY ; ACID ; TARGET ; PEPTIDES ; PROTEOMICS ; MS ; methods ; TECHNOLOGY ; FRAGMENT ; MIXTURES ; QUANTITATIVE PROTEOMICS ; STRATEGY ; TIMES ; SILAC ; Type ; ACCURATE QUANTITATION ; MIPA ; Sequence permutation
    Abstract: A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC-MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity
    Type of Publication: Journal article published
    PubMed ID: 20104620
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  • 7
    Abstract: An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit C beta 1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of C beta 1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes
    Type of Publication: Journal article published
    PubMed ID: 20564261
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  • 8
    Keywords: IONS ; DISSOCIATION ; PROTONATED PEPTIDES ; NOMENCLATURE ; PROTEIN IDENTIFICATION ; databases ; SPECTROMETER ; TAGS
    Abstract: The accurate mass values of all immonium, y(1), y(2), a(2), and b(2) ions of tryptic peptides composed of the 20 standard amino acids were calculated. The differences between adjacent masses in this data set are greater than 10 mDa for more than 80% of the values. Using this mass list, the majority of low mass ions in quadrupole-time of flight tandem mass spectra of peptides from tryptic digests and from an elastase digest could be assigned. Besides the a(2)/b(2) ions, which carry residues 1-2 from the N-terminus, a variety of internal dipeptide b ions were regularly observed. In case internal proline was present, corresponding dipeptide b ions carrying proline at the N-terminal position occurred. By assigning the dipeptide b ions on the basis of their accurate mass, bidirectional or unidirectional sequence information was obtained, which is localized to the peptide N-terminus (a(2)/b(2) ions) or not localized (internal b ions). Identification of the y(1) and y(2) ions by their accurate mass provides unidirectional sequence information localized to the peptide C-terminus. It is shown that this patchwork-type sequence information extractable from accurate mass data of low-mass ions is highly efficient for protein identification.
    Type of Publication: Journal article published
    PubMed ID: 11987126
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  • 9
    Keywords: KINASE ; PEPTIDES ; POSTTRANSLATIONAL MODIFICATIONS ; MS/MS ; ABSOLUTE QUANTIFICATION ; STOICHIOMETRY ; signaling networks ; multisite phosphorylation ; QUANTITATIVE PROTEOMICS ; LABELING STRATEGY
    Abstract: This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site-specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry-based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site-specific degrees of phosphorylation including the label-free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.
    Type of Publication: Journal article published
    PubMed ID: 22653803
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  • 10
    Keywords: PROTEINS ; PHOSPHORYLATION ; PERFORMANCE ; MASS-SPECTROMETRY ; ABSOLUTE QUANTIFICATION ; PROTON AFFINITIES
    Abstract: A novel type of peptide standard is introduced that consists of two peptides combined in one synthetic molecule and separated by a proteolytic cleavage site. Upon enzymatic digestion, the two peptides are released in a molar one-to-one ratio. This method enables the generation of exact equimolar mixtures of two peptides of any nature and origin, thereby providing a valuable tool for the investigation of fundamental phenomena in MS. The applicability of the method is exemplified by the analysis of the effect of peptide sequence variations on the relative ionization efficiency in ESI- and MALDI-MS.
    Type of Publication: Journal article published
    PubMed ID: 23090848
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