Sample preparation and digestion for proteomic analyses using spin filters

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

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“…Subsequent steps, i.e. carbamidomethylation, alkylation and tryptic digestion, were performed exactly as described before [47,48]. …”

Section: Methodsmentioning

confidence: 99%

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Ludwig

Miroschedji

Doeppner

et al. 2018

J of Extracellular Vesicle

175

206

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“…After elution in 0.1 M glycine (pH 3.5), proteins were dialyzed in 8 M urea, 50 mM NH 4 HCO 3 , 0.5 mM TCEP using 10-kDa cut-off microcon devices (Millipore) as published (20). Prior to digestion, the urea concentration was diluted to 1.5 M in 50 mM NH 4 HCO 3 , 0.5 mM TCEP.…”

Section: Methodsmentioning

confidence: 99%

Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway

Déléris

Trost

Topisirović

et al. 2011

Journal of Biological Chemistry

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Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinaseactivated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.ERK3 and ERK4 define a distinct subfamily of atypical MAP kinases that display structural and functional differences with conventional MAP kinases (1). First, they possess a single phospho-acceptor site (Ser-Glu-Gly) in their activation loop instead of the classical dual phosphorylation Thr-Xxx-Tyr motif (2, 3). Accordingly, they are not phosphorylated and activated by dual-specificity MAP kinase kinase family members (4, 5). Second, ERK3 and ERK4 bear the sequence Ser-Pro-Arg instead of Ala-Pro-Glu in subdomain VIII of the kinase domain, being the only kinases in the human kinome to have an arginine at this position. Third, contrary to conventional MAP kinases like ERK1/ERK2, which are multifunctional Ser/Thr kinases capable of phosphorylating a vast array of substrates, ERK3 and ERK4 appear to have a very narrow substrate specificity (6). Their only known physiological substrate is the protein kinase MK5 5 (7-10). The regulation of ERK3 and ERK4 activity remains poorly understood. We and others have shown that the serine residue within the activation loop of ERK3/ERK4 (Ser-189/Ser-186) is phosphorylated in vivo (6, 11-13). Catalytically inactive forms of the kinases are similarly phosphorylated on this motif, indicating that activation loop phosphorylation is mediated by an upstream cellular kinase (12, 13). Phosphorylation of Ser-189/Ser-186 leads to enzymatic activation of ERK3/ERK4 and is required for binding to and for cytoplasmic relocalization of the substrate MK5 (12, 13). Recently, we have show...

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“…The conventional sample processing methods in proteomics, namely SDS-PAGE, or in-solution-based sample processing, are slow and laborious and thus do not easily provide the reproducibility and throughput to meet current demands. A paradigm shift was the introduction of a filteraided sample processing method (FASP), which is initially described by Manza et al (1) and then fully realized in practice by Wisniewski et al (2). These filter-aided methods make use of ultrafiltration membranes with molecular weight cut offs (MWCO) in the 10 to 30 kDa range to efficiently remove small molecules and salts and to capture denatured proteins on a cellulose filter even if the molecular weight of the protein is much smaller than the nominal MWCO of the ultrafiltration membrane.…”

mentioning

confidence: 99%

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates

Berger

Ahmed

Muntel

et al. 2015

Molecular & Cellular Proteomics

107

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We describe a 96-well plate compatible membrane-based proteomic sample processing method, which enables the complete processing of 96 samples (or multiples thereof) within a single workday. This method uses a large-pore hydrophobic PVDF membrane that efficiently adsorbs proteins, resulting in fast liquid transfer through the membrane and significantly reduced sample processing times. Low liquid transfer speeds have prevented the useful 96-well plate implementation of FASP as a widely used membrane-based proteomic sample processing method. We validated our approach on whole-cell lysate and urine and cerebrospinal fluid as clinically relevant body fluids. Without compromising peptide and protein identification, our method uses a vacuum manifold and circumvents the need for digest desalting, making our processing method compatible with standard liquid handling robots. Mass spectrometry (MS)-based proteomics is moving increasingly into the translational and clinical research arena, where robust and efficient sample processing is of particular importance. The conventional sample processing methods in proteomics, namely SDS-PAGE, or in-solution-based sample processing, are slow and laborious and thus do not easily provide the reproducibility and throughput to meet current demands. A paradigm shift was the introduction of a filteraided sample processing method (FASP), which is initially described by Manza et al.(1) and then fully realized in practice by Wisniewski et al. (2). These filter-aided methods make use of ultrafiltration membranes with molecular weight cut offs (MWCO) in the 10 to 30 kDa range to efficiently remove small molecules and salts and to capture denatured proteins on a cellulose filter even if the molecular weight of the protein is much smaller than the nominal MWCO of the ultrafiltration membrane. Thus, the denaturation step is crucial to ensure that proteins much smaller than the nominal MWCO are efficiently retained by, e.g. a 10 kDa MWCO filter.In translational and clinical proteomics, which normally include large cohorts, the multititer-well plate is the preferred format for sample processing and storage. Although the application of FASP in the 96-well plate format has been described (3, 4), the major limitation of FASP in the 96-well plate is the much slower speed at which the 96-well plates have to be centrifuged: while a single ultrafiltration unit withstands up to 14,000 ϫ g, the 96-well plate format can only be centrifuged at g-forces of up to 2,200 ϫ g. This significantly lower g-force for 96-well plates results in a slow liquid transfer, which in turn considerably prolongs the required centrifugation times to hours instead of tens of minutes for the three to four necessary centrifugation steps (i) for the initial loading, reduction and alkylation, (ii) for the different washing steps, and (iii) for the elution (3).Independent of the format FASP is performed in, the conventional FASP also requires relative large volumes of high salt concentration for efficient elution of the tryptic peptid...

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Sample preparation and digestion for proteomic analyses using spin filters

Cited by 355 publications

References 11 publications

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway

MStern Blotting–High Throughput Polyvinylidene Fluoride (PVDF) Membrane-Based Proteomic Sample Preparation for 96-Well Plates

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