|Purification of Radiolabeled Oligonucleotides Using ZipTipC18 Pipette Tips|
|Mass Spectrometry Workflow Solutions|
|Reference overview||Application||Pub Med ID|
|A CROSS-SPECIES STUDY OF PI3K PROTEIN-PROTEIN INTERACTIONS REVEALS THE DIRECT INTERACTION OF P85 AND SHP2|
Susanne B. Breitkopf, Xuemei Yang, Michael J. Begley, Meghana Kulkarni, Yu-Hsin Chiu, Alexa B. Turke, Jessica Lauriol, Min Yuan, Jie Qi, Jeffrey A. Engelman, Pengyu Hong, Maria I. Kontaridis, Lewis C. Cantley, Norbert Perrimon, John M. Asara
Sci Rep. 2016
Using a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.
|FOOD PEPTIDOMICS OF IN VITRO GASTROINTESTINAL DIGESTIONS OF PARTIALLY PURIFIED BOVINE HEMOGLOBIN: LOW-RESOLUTION VERSUS HIGH-RESOLUTION LC-MS/MS ANALYSES|
Juliette Caron, Gabrielle Chataigne, Jean-Pascal Gimeno, Nathalie Duhal, Jean-Francois Goossens, Pascal Dhulster, Benoit Cudennec, Rozenn Ravallec, Christophe Flahaut
Consumers and governments have become aware how the daily diet may affect the human health. All proteins from both plant and animal origins are potential sources of a wide range of bioactive peptides and the large majority of those display health-promoting effects. In the meat production food chain, the slaughterhouse blood is an inevitable co-product and, today, the blood proteins remain underexploited despite their bioactive potentiality. Through a comparative food peptidomics approach we illustrate the impact of resolving power, accuracy, sensitivity, and acquisition speed of low-resolution (LR)- and high-resolution (HR)-LC-ESI-MS/MS on the obtained peptide mappings and discuss the limitations of MS-based peptidomics. From in vitro gastrointestinal digestions of partially purified bovine hemoglobin, we have established the peptide maps of each hemoglobin chain. LR technique (normal bore C18 LC-LR-ESI-MS/MS) allows us to identify without ambiguity 75 unique peptides while the HR approach (nano bore C18 LC-HR-ESI-MS/MS) unambiguously identify more than 950 unique peptides (post-translational modifications included). Herein, the food peptidomics approach using the most performant separation methods and mass spectrometers with high-resolution capabilities appears as a promising source of information to assess the health potentiality of proteins.
|Comparative evaluation of peptide desalting methods for salivary proteome analysis.|
Nico Jehmlich, Claas Golatowski, Annette Murr, Gesell Salazar, Vishnu Mukund Dhople, Elke Hammer, Uwe Volker
Clin Chim Acta 2014
|ISOLATION AND IDENTIFICATION OF MEMBRANE VESICLE-ASSOCIATED PROTEINS IN GRAM-POSITIVE BACTERIA AND MYCOBACTERIA|
Rafael Prados-Rosales, Lisa Brown, Arturo Casadevall, Sandra Montalvo-Quiros, Jose L. Luque-Garcia
Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicle-associated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are:<br />• Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant.<br />• The use of an ultrafiltration system, which allows concentrating larger volumes.<br />• In solution digestion of proteins followed by peptides purification by ziptip.
|Using theoretical protein isotopic distributions to parse small-mass-difference post-translational modifications via mass spectrometry.|
Timothy W. Rhoads, Jared R. Williams, Nathan I. Lopez, Jeffrey T. Morre, C. Samuel Bradford, Joseph S. Beckman
J Am Soc Mass Spectrom 2013
|DETERMINIG IN VIVO PHOSPHORYLATION SITES USING MASS SPECTROMETRY|
Susanne B. Breitkopf, John M. Asara
Curr Protoc Mol Biol 2012
Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems, since it controls cell growth, proliferation, survival, and other processes. High-resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity, and throughput. The protocols described here focus on two common strategies: (1) identifying phosphorylation sites from individual proteins and small protein complexes, and (2) identifying global phosphorylation sites from whole-cell and tissue extracts. For the first, endogenous or epitope-tagged proteins are typically immunopurified from cell lysates, purified via gel electrophoresis or precipitation, and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO(2)) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time consuming and involve digesting the whole-cell lysate, followed by peptide fractionation by strong cation-exchange chromatography, phosphopeptide enrichment by IMAC or TiO(2), and LC-MS/MS. Alternatively, the protein lysate can be fractionated by SDS-PAGE, followed by digestion, phosphopeptide enrichment, and LC-MS/MS. One can also immunoprecipitate only phosphotyrosine peptides using a pTyr antibody followed by LC-MS/MS.
|Rapid metabolite identification with sub parts-per-million mass accuracy from biological matrices by direct infusion nanoelectrospray ionization after clean-up on a ZipTip and LTQ/Orbitrap mass spectrometry.|
John C. L. Erve, William DeMaio, Rasmy E. Talaat
Rapid Commun Mass Spectrom 2008
Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100,000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments
|COMPARISON OF SDS- AND METHANOL-ASSISTED PROTEIN SOLUBILIZATION AND DIGESTION METHODS FOR ESCHERICHIA COLI MEMBRANE PROTEOME ANALYSIS BY 2-D LC-MS/MS|
Nan Zhang, Rui Chen, Nelson Young, David Wishart, Philip Winter, Joel H. Weiner, Liang Li
|Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS.|
Takashi Obama, Rina Kato, Yutaka Masuda, Katsuhiko Takahashi, Toshihiro Aiuchi, Hiroyuki Itabe
Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.
|GEL-FREE SAMPLE PREPARATION FOR THE NANOSCALE LC-MS/MS ANALYSIS AND IDENTIFICATION OF LOW-NANOGRAM PROTEIN SAMPLES|
Marco Gaspari, Vittorio Abbonante, Giovanni Cuda
J Sep Sci. 2007
Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 microm id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified protei
|Are ZipTip pipette tips compatible with robotic / automated sample preparation systems?||ZipTipm-C18 compatibility has been demonstarted with the following robotic / automated sample preparation systems:
1. ABI Symbiot™ Sample Workstation for MALDI TOF Biospectrometry™ with Universal Cone for ZipTip (cat. no. GEN602987) and Rack Adapter for the ZipTip box.
2. Bruker Daltonics MAP™ II and MAP™ II/8 MADLI Auto Prep Station
3. Micromass 2700-MS and MassPREP™ Sample Preparation Stations
4. Genomic Solutions ProMS™ Sample Preparation Station
5. Cy-Bio CyBi™ Well 96 automated pipettor
6. Packard BioScrences MultiPROBE
7. Tecan GENESIS Workstation
|Can ZipTip be reused?||ZipTip is intended for single use.|
|How quickly can I desalt and concentrate peptide, protein or oligonucleotide samples with ZipTip pipette tips?||ZipTip concentrates, and desalts samples in about one minute.|
|I've noticed that not all of my peptides were recovered when using ZipTipµ-C18. What should I do?||First, review the steps outlined in the Troubleshooting section of the Reversed-phase Operators Manual. Decreased recoveries may be due to either poor binding or insufficient elution of the sample from the resin. Increased concentration of Acetonitrile in the elution solution may improve recovery of highly hydrophobic peptide. If none of the suggested approaches work try ZipTipC18, the longer bed volume may improve peptide capture resulting in better recovery.|
|I am performing a step gradient elution with ZipTipC18, ZipTipµ-C18 or ZipTipC4. How can I eliminate carry over from one step in a gradient to the next?||To minimize sample carry-over when using a step gradient fractionation procedure, we recommend thoroughly washing the ZipTip with 3 cycles of the respective eluant prior to switching (moving) to the next gradient eluant. Example: Adsorbed sample was subjected to an initial step gradient elution with 10% acetonitrile. The tip should be immediately washed using 3 cycles of 10% acetonitrile to waste prior to subsequent gradient elution steps (i.e. stepping the elution gradient to 30% acetonitrile).|
|I don't want to analyze my mass spec product immediately after elution from ZipTip, can I store the eluted product safely for future analysis?||Yes, elute the product from ZipTip into a clean vial, close the vial and seal with parafilm. Store the wrapped vial at -20ºC until required for analysis.|
|What is the chemical compatibility of ZipTip?||ZipTip pipette tips are compatible with most commonly used solvents used in protein structural analysis. See the detailed chemical compatibility chart located in the ZipTip Operators Manuals.|
|Can I use higher than 50% ACN to elute my sample from ZipTipC18 and ZipTipm-C18?||Yes, however the tip should be prewet 5 times with 10 ml of 100% ACN followed by a wash of 10 ml 0.1% TFA. Then bind, wash and elute.|
|After elution from ZipTip my spots won't dry. Want can I do?||For the initial prewet step wash the tip 5 times with 100% ACN followed by 0.1% TFA. Then bind, wash and elute as normal.|
|Can I use ZipTipC18 or ZipTipµ-C18 for amino acids?||ZipTipC18 and ZipTipµ-C18exhibit comparable binding properties to C18 reversed-phase HPLC. As such, lower recoveries will be seen for hydrophilic amino acids.
C18 is offered in two bed volumes:
ZipTipC18 - a standard bed of 0.6 µL for sample elution in 1 to 4 µL
ZipTipµ-C18 - a micro bed of 0.2 µL for sample elution in < 1 µL.
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