A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Summary

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Abstract

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Introduction

For decades, surgically excised human tissues have been archived as formalin-fixed, paraffin-embedded (FFPE) blocks. These tissue blocks initially preserve the three-dimensional (3D) structure embedded in paraffin. The tissues are subsequently sliced using microtomes, mounted onto slides, and stained-commonly with hematoxylin and eosin (HE) or immunohistochemistry (IHC)-to facilitate histopathological diagnosis by experienced pathologists^1,2. FFPE tissues offer distinct advantages for long-term storage due to the protein crosslinking induced by formalin, which halts enzymatic and proteolytic activity

Protocol

This study was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (IRB No. 1904-114-1028). All participants provided written informed consent to participate in the study. Detailed information on all materials used in this protocol is presented in the Table of Materials.

1. FFPE tissue antigen retrieval for proteomics sample preparation

NOTE: Ensure that the scalpels and all materials, such as the tube used, are sterile to avoid any cross-contamination. Protocols of this study can be adapted for any FFPE tissue with minor modifications base....

Results

The established suspension trapping filter-based proteomics sample preparation, combined with label-free quantitation using single-shot data-independent acquisition, were applied to pancreatic cystic FFPE tissues (Figure 1). Precise ROI isolation during the FFPE tissue processing was achieved across different pancreatic cystic FFPE tissues (Figure 2A), resulting in the acquisition of reproducible total ion chromatograms between biological tri-replicates for each.......

Discussion

This protocol outlines a rapid and efficient proteomics method that utilizes ROIs isolated from FFPE tissue sections mounted on glass slides for pathological diagnosis. When surgical intervention is advantageous, solid neoplasms such as cancers and cysts are surgically resected and preserved for pathological evaluation. For long-term storage, tissues are fixed in formalin and embedded in paraffin (FFPE). FFPE tissue blocks are then sectioned to a thickness of 4-10 µm, mounted on glass slides, subjected to antigen re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1% FA in ACN (LC-MS grade)	Fisher Chemical	LS120-212
0.1% FA in Water (LC-MS grade)	Fisher Chemical	LS118-4
0.5M TCEP	Thermo Scientific	77720
10% SDS	Invitrogen	2679093
1M TEAB (pH 8.5)	Sigma-Aldrich	102545001
1M Tris-cl (pH 8.5)	BIOSOLUTION	BTO21
A-14C centrifuge	Satorious	167709
Acetone (HPLC grade)	Fisher Scientific	A949-4
ACN (HPLC grade)	J.T.Baker	9017-88
CHCl3 (HPLC grade)	Thermo Scientific	022920.k2
CR paper	ADVANTEC	70406001
DIA-NN ver 1.9	Open source	https://github.com/vdemichev/DiaNN	Proteomics Search Engine
EPOCH2 microplate reader	Agilent	2106208
Ethanol	MERCK	K50505283 836
FA (LC-MS grade)	Fisher Chemical	A117-50
Ingenuity Pathway Analysis (IPA)	QIAGEN	830018	Bioinformatics tool
Lyophilizer (SRF110R+vaper trap)	Thermo Scientific	SRF-110-115
MeOH (HPLC grade)	MERCK	UN1230
Microplate BCA protein Assay kit-Reducing Agent Compatible	Thermo Scientific	23252
MSConvert	Open source	http://proteowizard.sourceforge.net/tools.shtml	MS data transformation software
Orbitrap Exploris 480	Thermo Scientific	MA10813C	MS
PepMAP RSLC C18 separation column	Thermo Scientific	ES903
Perseus	Open source	https://cox-labs.github.io/coxdocs/perseus_instructions.html	Statistical tool
PIERCE chloroacetamide No-Weigh Format	Thermo Scientific	A39270
PIERCE Quantitative colorimetric peptide Assay	Thermo Scientific	23275
Plate shaker	Green SSeriker	VS-202D
Probe sonicator	VibraCellTM	VCX750
Protein LoBind Tube 1.5 mL	Eppendorf	22431081
QSP 10 µL pipette Tip	Thermo Scientific	TLR102RS-Q
QSP 300 µL pipette Tip	Thermo Scientific	TLR106RS-Q
Scalpel	Bard-Parker	372615
S-Trap: Rapid Universal MS sample Prep	PROTIFI	CO2-mini-40
SureSTART Vial 0.2 mL	Thermo Scientific	6pk1655
TFA	Sigma-Aldrich	102614284
ThermoMixer C	Eppendorf	5382
Trypsin/Lys-C (LC-MS grade)	Promega	V5073
Vanquish NEO	Thermo Scientific	8348249	LC
Water (HPLC grade)	Honeywell	AH365-4
Xcalibur ver 4.7	Thermo Scientific	30966	MS data acquisition software
Xylene	Sigma-Aldrich	102033629