In-Depth Mass Spectrometry-Based Proteomics of Formalin-Fixed, Paraffin-Embedded Tissues with a Spatial Resolution of 50–200 μm

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 μm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 μm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.

Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics

“…However, for cases in which microscopy of isolated cells or tissues is a necessary part of the workflow, optically transparent glass nanowells remain preferable. Cases of such use include visual inspection of cell isolation or tissue collection following laser microdissection …”

“…Cases of such use include visual inspection of cell isolation or tissue collection following laser microdissection. 46…”

Slice-PASEF: fragmenting all ions for maximum sensitivity in proteomics

“…Recent progress in mass spectrometry instrumentation and data processing methods has led to a rapidly increasing interest in novel technologies and applications that involve proteomic profiling of ultra-low peptide amounts. These facilitate, for example, single-cell proteomics or spatial proteomic profiling of tissues, which can offer unique biological and biomedical insights [13][14][15][16][17][18][26][27][28][29] . Still, so far these methods have inevitably required peptide separation using nanoflow gradients to achieve the required sensitivity, including, in some cases, flow rates below 100 nl/min.…”

“…Large-scale proteomic screens for systems biology experiments and biomarker discovery in patient cohorts are now streamlined [3][4][5][6][7][8][9][10][11][12] . Furthermore, recent high-sensitivity applications, such as single-cell proteomics and spatial tissue proteomics, have been rapidly gaining traction in the field [13][14][15][16][17][18] . High sensitivity applications in proteomics are currently hampered by two key limitations.…”

Chemical Decrosslinking-Based Peptide Characterization of Formaldehyde-Fixed Rat Pancreas Using Fluorescence-Guided Single-Cell Mass Spectrometry

“…HIAR has been widely used in immunohistochemistry to improve the ability to detect antigens via restoring access to binding sites and their proper spatial confirmations. Many successful cases using heat treatment were shown in the spatial characterization of protein assisted by various recent techniques including a nanodroplet in a pot system. , Heating the fixed sample in acidic or basic buffer partially removes the crosslinks and often facilitates subsequent trypsinization needed for MS-based proteomics/peptidomics investigation of tryptic fragments. − …”