Evaluation of Fast and Sensitive Proteome Profiling of FF and FFPE Kidney Patient Tissues

Abstract

The application of proteomics to fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) human tissues is an important development spurred on by requests from stakeholder groups in clinical fields. One objective is to complement current diagnostic methods with new specific molecular information. An important goal is to achieve adequate and consistent protein recovery across and within large-scale studies. Here, we describe development of several protocols incorporating mass spectrometry compatible detergents, including Rapigest, PPS, and ProteaseMax. Methods were applied on 4 and 15 μm thick FF tissues, and 4 μm thick FFPE tissues. We evaluated sensitivity and repeatability of the methods and found that the protocol containing Rapigest enabled detection of 630 proteins from FF tissue of 1 mm2 and 15 μm thick, whereas 498 and 297 proteins were detected with the protocols containing ProteaseMax and PPS, respectively. Surprisingly, PPS-containing buffer showed good extraction of the proteins from 4 μm thick FFPE tissue with the average of 270 protein identifications (1 mm2), similar to the results on 4 μm thick FF. Moreover, we found that temperature increases during incubation with urea on 4 μm thick FF tissue revealed a decrease in the number of identified proteins and increase in the number of the carbamylated peptides.

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

“…There can be anywhere between 47-92% overlap of protein identification between FF and FFPE tissues 32,37,38 . Previous studies have sought to optimise individual steps in the sample preparation workflow, notably the deparaffinisation or the tissue solubilisation steps for FFPE samples 12,39 , the LC settings 40 or the type of MS fragmentation 15 .…”

Hanging-droplet sample preparation improves sensitivity of spatial proteomics

“…2F). Compared with previous spatially resolved proteomic studies of LCM tissues 26,28 in which at least 0.5 mm 2 sized tissues were required to quantify >1000 proteins, 23,46 the LCM-HD-nanoPOTS system provided > 1000 quantifiable proteins from 0.01 mm 2 sized tissues, i.e. using a ~50-fold smaller tissue volume.…”

“…47,48 To identify an optimal surfactant for spatial proteomics, we evaluated three commercially available and MScompatible surfactants, including DDM, ProteaseMax, and RapiGest (Table S2). RapiGest and ProteaseMax are widely used in low-input bottom-up proteomics studies, 46,49 as all have been demonstrated to improve protein solubility and tryptic digestion efficiency. They are also readily degraded under acidic condition, and thus are compatible with a single-tube preparation workflow.…”

Hanging drop sample preparation improves sensitivity of spatial proteomics

“…2F). Compared with other spatially resolved proteomic studies of LCMderived tissues, 23,46 in which at least 0.5 mm 2 sized tissues were required to quantify >1000 proteins, the LCM-HD-nanoPOTS system provided >1000 quantifiable proteins from 0.01 mm 2 sized tissues, i.e. using a ∼50-fold smaller tissue volume.…”

“…47,48 To identify an optimal surfactant for spatial proteomics, we evaluated three commercially available and MS-compatible surfactants, including DDM, ProteaseMax, and RapiGest (Table S2 †). RapiGest and ProteaseMax are widely used in low-input bottom-up proteomics studies, 46,49 as all have been demonstrated to improve protein solubility and tryptic digestion efficiency. They are also readily degraded under acidic condition, and thus are compatible with a single-tube preparation workflow.…”