|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
|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.
|How are ZipTip pipette tips packaged?||ZipTip pipette tips are available in four pack sizes:
|How can I elute from ZipTip pipette tips directly into a nanospray needle?||We suggest cutting a gel loading tip (Eppendorf 0030 001 222) approximately 2-3 mm above where the tip widens from the capillary end. ZipTip binding, washing and elution are performed utilizing the standard protocols listed in the ZipTip Reversed-Phase Operating Manual. Prior to the final dispense (elution of the peptide or protein) the cut down gel loading tip is pressed on to the end of the ZipTip using a slight twisting motion. The friction fit is tight, allowing sample to be dispensed directly into an nanospray needle.
Refer to the ZipTip Reversed-Phase Operators Manual for a detailed diagram demonstrating the above procedure.
|How can I improve salt removal with ZipTipC18, ZipTipµ-C18 or ZipTipC4?||Wash ZipTip with 3 to 5 aspirate/dispense cycles of 0.1% TFA (10 ml). If salt contamination persists, consider the following: For Peptides - Try washing peptides with 10% ACN (acetonitrile)/0.1% TFA. For proteins, the above ACN concentration can be increased to 30% Add 100 microliters of additional wash solution to a pipet and slowly push this solution through the Zip Tip. If the problem persists try blotting the Zip Tip on a paper towel to remove the residual wash soulution remainig in the Zip Tip after dispensing the final wash.|
|I'm performing electrospray / nanospray mass spec on my eluted sample from ZipTip. What is the appropriate solution to elute my sample with?||Peptides - 50% MeOH/0.1% TFA or 0.1% formic acid in Milli-Q water Proteins - 75% MeOH/0.1% TFA or 0.1% formic acid in Milli-Q water|
|I have a polypeptide that has a molecular weight greater than 50,000 Da. Which ZipTip should I use?||ZipTipC18 or ZipTipµ-C18 is most applicable for low molecular weight proteins and peptides (up to 20,000 Da) while ZipTipC4 is most suitable for low to intermediate molecular weight proteins (3,000 - 100,000 Da). In many cases, the two devices could be used interchangeably. Because higher molecular weight proteins tend to adsorb tenaciously to hydrophobic surfaces the ZipTipC4 is recommended for proteins over 100,000 Da.|
|I have detergent in my sample; will I be able to enrich my peptide digest with ZipTips?||Detergents will bind and concentrate with the peptides when using Reverse Phase tips such as ZipTipC18. Non-ionic and Zwitterionic detergents such as Tween-20, Chaps and triton cna be removed by ZipTipSCX at concentrations up to 0.1%. Ionic detergents such as SDS cannot be removed by ZipTips.|
|I have pipetted my sample onto the ZipTip. During the elution step, an air bubble was introduced into the tip. Will this decrease the recovery of peptides?||ZipTip is equipped with microchannels which travel up through the resin bed. Any air that is inadvertently introduced to the ZipTip can easily be flushed out.|
|I would like to separate a peptide from a protein. Is it possible to fractionate using ZipTip?||Yes, this can be accomplished by utilizing the step gradient elution protocol found in the ZipTip Reversed-Phase Operators Manual or Technical Note# TN226 entitled "Fractionation of Complex Peptide or Protein Mixtures Prior to MALDI-TOF MS Using ZipTipC18, ZipTipµ-C18 and ZipTipC4 Pipette Tips". The protocol includes multiple elution steps with increasing concentrations of acetonitrile in the elution buffer (e.g. 10%, 30%, 50%). The smaller molecules (weak binders) are eluted off the resin first and the larger molecules (strong binders) are eluted last.|
|Is it possible to use plasma with Zip Tip prior to mass spec?||Yes. The plasma needs to be diluted 1:4 and acidified (for example, a 1:1:2 mix of serum: 10% TFA : water).|