MSACL 2016 US Abstract

Tip-based Fractionation for Comprehensive Phosphoproteome Analysis

Alireza Dehghani (Presenter)
University of Bonn

Bio: I received my bachelor's degree in Cell and Molecular Biology at Shiraz University, Iran. After my graduation, I competed in a nationwide Master’s Degree Examination and I ranked 42nd among 12814 participants in the field of Biology. For my master studies, I enrolled in the Genetics program at Shahid Beheshti University, Tehran and I have 2 papers published from my Master’s thesis. As I was interested in proteomics related studies, I planned to do a Ph.D. in this field in a reputed institute which is equipped with modern facilities in the field of proteomics, especially mass spectrometry. I chose Germany for pursuing my higher education because it is one of the leading countries in the field of research and technology. I applied for a Ph.D. position at the Institute of Biochemistry and Molecular Biology (IBMB) in University Hospital Bonn and I got the position and later I won a German Academic Exchange Service full scholarship (DAAD) for my Ph.D. My Ph.D. thesis is entitled “Identification of Novel Proteins and Protein Networks Involved in the Differentiation of Multipotent Neural Progenitor Cells to Oligodendrocyte Precursor Cells”. My project involves evaluating promising candidate proteins which are possibly involved in this differentiation processes. The candidate proteins are Double Cortin Like Kinase 1 and Peptidyl Arginine Deiminase 2. We are investigating the role of these proteins in the differentiation from mulitpotent neural progenitor cells to oligodendrocyte precursor cells by knock down and over expression experiments. Moreover mass spectrometry based characterization and biochemical evaluations are performing including posttranslational modification analysis, identification of interaction partners and identification ofsubstrates in order to investigate mechanistic details of their involvement in the differentiation processes.

Authorship: Alireza Dehghani, Markus Gödderz, Dominic Winter
University of Bonn, Bonn, Germany

Short Abstract We developed a new workflow for phosphoproteomics studies which is able to find a considerable number of phosphosites with reduced time, cost and effort. The workflow is a combination of Titanium dioxide (TiO2) phosphopeptide enrichment followed by pipette tip-based Strong Cation-Exchange (SCX) fractionation. Using this method more than 9000 phosphosites and 7500 phosphopetides were detected out of 3 mg of HeLa lysate. The new workflow was successfully applied for the first time for the analysis of the effects of inhibition of cholesterol egress from lysosomes using U18666A inhibitor on the phosphoproteome of Mouse Embryonic Fibroblasts cells (Mef cells). 12881 phosphosites were found in this experiment which 751 were significantly regulated (349 phosphosites were up-regulated and 402 down-regulated).

Long Abstract

Phosphorylation is one of the most important and most abundant post translational modifications (PTMs). Phosphorylation involves adding a phosphate group to serine, tyrosine and threonine residues of a protein leading to a change of protein structure [1]. It has a major role in many cell functions such as protein-protein interactions, signal transduction and differentiation [2]. Therefore using phosphoproteome analysis and quantification of different cells and different tissues in different conditions has an important role in the identification of cellular mechanisms. However it is not easy to find the phosphopeptides among complex mixture of digested peptides. Advanced chromatography devices and phosphopeptide enrichment methods such as antibody immunoaffinity [3], Strong Cation Exchange (SCX) chromatography [4], Immobilized Metal Affinity Chromatography (IMAC) [5], Titanium dioxide TiO2 phosphopeptide enrichment [6] and Sequential Elution from IMAC (SIMAC) [7] paved the way for identifying great amount of phosphosites in recent years. As it was mentioned before, one of the obstacles in phosphoproteomics is the complexity of the samples, different fractionation methods such as SDS-PAGE [4], ion exchange [8], Hydrophilic Interaction Liquid Chromatography(HILIC) [9] and high pH RP chromatography [10] have used to tackle this problem. Fractionation can be done either before phosphopeptide enrichment or after it, although most scientists prefer to do it before enrichment [11]. Phosphoproteomic studies are usually time-consuming and expensive since they require large sample amounts and liquid chromatography-based separation followed by long run times on mass spectrometer instrument, so just limited laboratories around the world do large phosphoproteomics experiments. Here we established a simple, time-saving and affordable workflow, with the ability to find considerable numbers of phosphopeptides. This workflow is a combination of double TiO2 phosphopeptide enrichment followed by tip-based SCX fractionation. Tip-based fractionation is based on the StageTips method first described by Rappsilber and colleagues [12]. StageTips were prepared using a small amount of Empore material (3M) fitted into 200 μl micropipette tips. This tip-based method has several advantages such as ease of use, flexibility, reproducibility and low-cost [13]. HeLa lysate in 3 mg aliquots were prepared for the following experiments. Phosphopeptide enrichment was done for each aliquot using double TiO2 enrichment and samples were desalted with OASIS HLB cartridges. Phosphopeptides were fractionated to 6 fractions using the following methods: pipette tip-based strong cation-exchange (SCX), pipette tip-based strong anion-exchange (SAX) and pipette tip-based concatenated high-pH reversed-phase. Fractions from different methods were desalted using C18 StageTips. Peptides were resuspended in 5% acetonitrile, 5% formic acid solution and analyzed using an EASY-nLC 1000 UHPLC system coupled to an OrbitrapVelos mass spectrometer. Each fraction was analyzed by 120 min LC-MS/MS gradients using Multi-Stage Activation (MSA). The best results among the pipette tip-based methods studied were obtained from SCX fractionation which provided more than 9000 phosphosites, following with the concatenated high-pH reversed-phase and SAX that more than 6000 and 4000 phosphosites were detected respectively using each method. Phosphopeptides also were analyzed without fractionation using 240 min LC-MS/MS gradients which 2690 phophosites were found. Pipette tip-based SCX method was also compared with the SCX chromatography which is the standard method for large-scale phosphoproteomics studies [14][15]. 3 mg of Hela lysate were fractionated to 12 fractions using an ÄKTA purifier system equipped with a Polysulfoethyl A column. Afterwards, phosphopeptide enrichment was done for each fraction separately using double TiO2 method. These fractions were analyzed using 90 min LC-MS/MS gradients, and more than 10000 phosphopeptides were detected.

The established workflow was used for the analysis of the effects of U18666A inhibitor on the phosphoproteome of Mouse Embryonic Fibroblasts cells (Mef cells). U18666A is a well established drug commonly used to phenocopy mutations in the NPC1 gene by inhibiting cholesterol trafficking among lysosomes and other cell compartments resulting in cholesterol accumulation in lysosomes [16]. SILAC-treated MEF cells (Arg-10 and Lys-8) were incubated for 24 hr with the U18666A (3µg per ml), as control MEF cells (Arg-0, Lys-0) were treated with DMSO. Cholesterol accumulation in lysosomes was verified by Filipin staining. Same amount of the sample lysate and control (3 mg each) were combined and digested using trypsin after reduction and alkylation. Double TiO2 phosphopeptide enrichment and tip-based SCX fractionation was done for the digested peptides as it was explained before. In order to be able to monitor the changes in phosphoproteome, whole cell lysate was run on SDS-PAGE and fractionated to 10 fractions and analyzed using mass spectrometry. 12881 phosphosites were found in this experiment which 751 were significantly regulated (349 phosphosites were up-regulated and 402 down-regulated).

In this study we have applied a new workflow (double TiO2 enrichment prior to pipette tip-based SCX fractionation) for the phosphoproteome analysis of cell lysates. With this new workflow we could find a considerable amount of phosphopeptides which is comparable with the standard approach for phosphoproteomic studies (SCX chromatography followed by phosphopeptide enrichment). However, the new workflow has many advantages compared to the common methods. It is so much simpler, and it also has a time-saving protocol and does not need any chromatography instrument. Using this method many laboratories will be able to do large phosphoproteomics experiment in a decent amount of time and also clinical laboratories can use it for clinical diagnostics.

References & Acknowledgements:

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[11] K. Engholm-keller and M. R. Larsen, “Technologies and challenges in large-scale phosphoproteomics,” Proteomics, vol. 13, pp. 910–931, 2013.

[12] J. Rappsilber, Y. Ishihama, and M. Mann, “Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption / Ionization , Nanoelectrospray , and LC / MS Sample Pretreatment in Proteomics,” Anal. Chem., vol. 75, no. 3, pp. 663–670, 2003.

[13] J. Rappsilber, M. Mann, and Y. Ishihama, “Protocol for micro-purification , enrichment , pre-fractionation and storage of peptides for proteomics using StageTips,” Nat. Protoc., vol. 2, no. 8, pp. 1896–1906, 2007.

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[16] R. J. Cenedella, “Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes.,” Lipids, vol. 44, no. 6, pp. 477–87, Jun. 2009.

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