SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics

Abstract

Large scale proteomic profiling of cell lines can reveal molecular signatures attributed to variable genotypes or induced perturbations, enabling proteogenomic associations and elucidation of pharmacological mechanisms of action. Although isobaric labeling has increased the throughput of proteomic analysis, the commonly used sample preparation workflows often require time-consuming steps and costly consumables, limiting their suitability for large scale studies. Here, we present a simplified and cost-effective one-pot reaction workflow in a 96-well plate format (SimPLIT) that minimizes processing steps and demonstrates improved reproducibility compared to alternative approaches. The workflow is based on a sodium deoxycholate lysis buffer and a single detergent cleanup step after peptide labeling, followed by quick off-line fractionation and MS2 analysis. We showcase the applicability of the workflow in a panel of colorectal cancer cell lines and by performing target discovery for a set of molecular glue degraders in different cell lines, in a 96-sample assay. Using this workflow, we report frequently dysregulated proteins in colorectal cancer cells and uncover cell-dependent protein degradation profiles of seven cereblon E3 ligase modulators (CRL4 CRBN ). Overall, SimPLIT is a robust method that can be easily implemented in any proteomics laboratory for medium-to-large scale TMT-based studies for deep profiling of cell lines.

A Degron Blocking Strategy Towards Improved CRL4CRBN Recruiting PROTAC Selectivity**

“…To set‐up our design principles, we focused on CC‐885 ( 1 ) as a proof of concept (Figure 1a). CC‐885 was shown to have broad substrate specificity, inducing the degradation of both Zinc Finger (ZF) containing and non‐ZF containing neo‐substrates [23,24] . Furthermore, detailed structural data of the DDB1‐CRBN‐CC885‐GSPT1 complex are publicly available [23] .…”

“…CC-885 was shown to have broad substrate specificity, inducing the degradation of both Zinc Finger (ZF) containing and non-ZF containing neosubstrates. [23,24] Furthermore, detailed structural data of the DDB1-CRBN-CC885-GSPT1 complex are publicly available. [23] Initial comparison of this GSPT1 structure to that of the lenalidomide induced CK1α ternary complex (Supplementary Figure 1a) suggests little similarity between the neo-substrate contact points to CRBN.…”

“…Sample preparation: Samples for quantitative proteomics were prepared using the SimPLIT workflow. [24] Briefly, the cell pellets were lysed in a buffer containing 150 μL 0.1 M triethylammonium bicarbonate (TEAB), 1 % sodium deoxycholate (SDC), 10 % isopropanol, 50 mM NaCl, 5 mM tris-2-carboxyethyl phosphine (TCEP), 10 mM iodoacetamide (IAA), universal nuclease, and supplemented with protease and phosphatase inhibitors. Samples were bath sonicated for 5 min, incubated at RT for 45 min and protein concentration was measured with the Bradford Protein Assay (Biorad) according to manufacturer instructions.…”

Isobaric labeling: Expanding the breadth, accuracy, depth, and diversity of sample multiplexing

“…The hardware and software innovations mentioned above are complimented by vigorous and reproducible sample preparation strategies that aim to improve proteome depth and overall throughput. Numerous sample preparation strategies for isobaric labeling experiments are available, with some geared toward specific applications [37][38][39][40][41][42][43][44][45]. The streamlined TMT (SL-TMT) protocol [46], for example, was designed with the intent to establish a highly standardized workflow that minimizes the steps needed for efficient and reproducible sample preparation for TMT-based experiments.…”

Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour

“…Several studies have described sample preparation methods for MS that enable highthroughput workflows, unfortunately most are typically developed for easy-to-lyse samples like mammalian cell lines or biofluids such as cerebral spinal fluid, plasma, or urine [1][2][3][4] . Solid tissue punches or sections and difficult-to-homogenise tissues like muscle and formalin-fixed tissues present greater solubilisation and protein extraction challenges [5][6][7] .…”