Dynamic Organellar Maps for Spatial Proteomics

Abstract

Eukaryotic cells are highly compartmentalized and protein subcellular localization critically influences protein function. Identification of the subcellular localizations of proteins and their translocation events upon perturbation has mostly been confined to targeted studies or laborious microscopy-based methods. Here we describe a systematic mass spectrometry-based method for spatial proteomics. The approach uses simple fractionation profiling and has two applications: Firstly it can be used to infer subcellular protein localization on a proteome-wide scale, resulting in a protein map of the cell. Secondly, the method permits identification of changes in protein localization, by comparing maps made under different conditions, as a tool for unbiased systems cell biology. C 2018 by John Wiley & Sons, Inc.Keywords: mass spectrometry r organelle proteomics r protein dynamics r spatial proteomics r subcellular localization r systems biology Figure 1 Overview workflow for the generation and analysis of organellar maps. See main text for details. Abbreviations: ER, endoplasmic reticulum; SVM, support vector machine; SILAC, stable isotope labeling by amino acids in cell culture; MR, movement reproducibility; PCA, principal component analysis; FDR, false discovery rate.support vector machine-based classification (see Basic Protocol 8), and assignment of protein copy numbers (see Basic Protocol 9). STRATEGIC PLANNINGCells are fractionated by six differential centrifugation steps into six pellets and one supernatant, followed by identification and quantification of the proteins in each fraction. The method does not result in purified organelles but uses the distribution profiles across the differential centrifugation fractions to infer protein localization via comparison with Itzhak et al. of 29Current Protocols in Cell Biology 4. With cells filled in the open syringe, introduce the plunger and force cell suspension past the bore five times in total. Take care not to introduce air into the device.Itzhak et al.

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“…Many if not most cell biological processes are accompanied by protein subcellular localisation changes (Lundberg and Borner, 2019). Hence, we adapted our previously developed method for generating organellar maps, to pinpoint the subcellular localisations of thousands of proteins in a single experiment (Itzhak et al, 2017; 2018). The comparison of organellar maps made under different physiological conditions allows the capture of drug induced protein translocations (Itzhak et al, 2016), and thus provides a universal and scalable tool for inferences about cellular responses and drug targets.…”

“…Protein concentrations were estimated by BCA assay. Equal amounts of SILAC heavy reference fraction were mixed with each SILAC light subfraction, acetone precipitated and subjected to in-solution digest and stage-tip peptide cleanup as described (Itzhak et al, 2018), prior to mass spectrometric analysis.…”

“…Next, only proteins detected in at least two replicates were retained (7427 in total). Copy number estimates were calculated using the Proteomic Ruler (Wiśniewski et al, 2014), as implemented in Perseus software (.5, (Tyanova et al, 2016), and described in (Itzhak et al, 2018). Protein intensities were scaled to molecular mass.…”

“…Generation of organellar maps and outlier testing followed the principles described in detail (Itzhak et al, 2018; 2016), with some modifications to accommodate a comparison across three conditions. Only proteins with high quality SILAC ratios in all 30 subfractions, i.e.…”

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“…We previously generated six organellar maps from HeLa (duplicates of wild-type, AP4B1 knockout, and AP4E1 knockout; Davies et al, 2018), and analysed them with our established MR (movement and reproducibility) test (Itzhak et al, 2019) to identify proteins with altered subcellular localisation. Briefly, for each protein, the abundance profiles from wild-type and knockout maps were subtracted pairwise, to obtain four sets of 'delta profiles' (wild-type-AP4B1, wild-type-AP4E1, both in duplicate).…”