Unlock the Full Potential of FFPE Tissue in Proteomics
App Note / Case Study
Last Updated: September 18, 2023
(+ more)
Published: April 28, 2023
Credit: Preomics
Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable resource to study various diseases and aid biomarker discovery.
Yet, despite its potential, scientists often refrain from using FFPE tissue in mass spectrometry (MS)-based proteomic studies as sample preparation is technically demanding and often unsuitable for the analysis of large cohorts.
This application note highlights how combining the BeatBox tissue homogenizer with PreOmics‘ iST sample preparation technology overcomes these challenges.
Download this App Note to:
- Discover a new method of FFPE sample preparation for LC-MS-based proteomics using the BeatBox tissue homogenizer
- Learn how you can process up to 96 samples per day
- Optimize your reproducibility while minimizing hands-on-time
PreOmics Application Note – FFPE sample preparation simplified with BeatBox: Version 1.0 - For research use only Page | 1 of 5
A xylene-free, high-throughput workflow for in-depth tissue proteome analysis
FFPE sample preparation simplified with BeatBox:
A xylene-free, high-throughput workflow for
in-depth tissue proteome analysis
Keywords
Proteomics, FFPE, BeatBox, highthroughput sample preparation, iST
technology, HR-mass spectrometry,
LC-MS
Key takeaways
• Streamlined FFPE sample preparation
workflow combining BeatBox and iST
technology
• High-throughput processing of up to
96 samples/day to ready-to-measure
peptides
• Optimized xylene-free approach
without deparaffinization for LC-MSbased proteomics
• Fast, easy-to-use, and standardized
protocol for high levels of reproducibility
and reduced hands-on time
• BeatBox workflow performance with
FFPE samples is equivalent to working
with fresh frozen tissue
Application Note
Authors: Katharina Limm, Silvia Würtenberger, Marcello Stein, Nils A. Kulak and Katrin Hartinger
PreOmics GmbH, Planegg/Martinsried, Germany
Introduction
Formalin-fixed, paraffin-embedded (FFPE)
tissue is an invaluable resource for retrospective studies to investigate molecular mechanisms or to discover novel biomarkers.1
Since
clinical FFPE specimens are collected and
stored routinely by hospital pathology departments, large libraries that are linked to clinical
metadata are readily available.2,3
FFPE tissue sample preparation for proteomic analyses is extremely challenging as formalin-fixation makes proteins difficult to extract.
Harsh conditions must be applied to reverse
protein cross-linking.2 In addition, paraffin interferes in the downstream liquid chromatography-mass spectrometry (LC-MS) analysis
which is why most protocols include an initial
xylene-based deparaffinization step that is
time-consuming, poses a health hazard due
to high levels of toxicity, and results in low reproducibility with the risk of sample loss.4,5
We present an optimized solution using
the BeatBox tissue homogenizer and iST
proteomic sample preparation technology
that simplifies, speeds-up and standardizes
FFPE sample preparation (Fig. 1). The workflow
eliminates the need for xylene-based
deparaffinization, and enables fast and robust
processing of up to 96 samples in parallel
from starting material to clean peptides
within one working day. We benchmarked the
BeatBox FFPE workflow against a traditional
sonication approach and evaluated its
effectiveness for deep proteomic analysis of
FFPE tissue in comparison with fresh frozen
tissue.
Figure 1 | Overview of the BeatBox FFPE workflow. High-throughput LC-MS-based proteomics workflow for FFPE tissue without xylenebased deparaffinization using the BeatBox platform in combination with optimized iST sample preparation including additional washing steps.
Tissue
homogenization
Protein extraction
Sample
BeatBox
Extracted protein Digest & purify LC-MS analysis
iST kit
+Additional washing steps (WASH 0)
De-crosslinking
Thermoshaker
PreOmics Application Note – FFPE sample preparation simplified with BeatBox:
A xylene-free, high-throughput workflow for in-depth tissue proteome analysis
Version 1.0 - For research use only Page | 2 of 5
Methods *for protocol details please see reference 6
Tissue samples
Studies were performed using FFPE as well as fresh frozen
mouse cardiac muscle, kidney and liver tissue provided by the
Research Institute of Molecular Pathology in Vienna and the
Histology Facility at Vienna BioCenter Core Facilities (Austria).
For the experiments, 10 µm FFPE tissue “full” curls containing
paraffin (approximately 37, 78, and 143 mm2 tissue area for
cardiac muscle, kidney, and liver, respectively), deparaffinized
FFPE tissue from “full” curls of the same size, and 1-2 mg of
fresh frozen tissue were compared. A xylene-based protocol
was employed for deparaffinization.
Sample preparation
Table 1 provides an overview of all sample preparation
workflows. FFPE samples (“full” curls or tissue deparaffinized
with xylene) were homogenized either with BeatBox (10 min,
HIGH setting, with BeatBox Tissue Kit 96x) or with beadbased sonication (10 cycles, 30 sec on, 30 sec off, 1.5 mL
tubes).7 Fresh frozen tissue was only homogenized using the
BeatBox (10 min, STANDARD setting, with BeatBox Tissue Kit
96x).
After homogenization, FFPE samples were incubated at
95°C to de-crosslink and solubilize protein. Subsequently, all
FFPE tissue Fresh frozen tissue
Xylene-based deparaffinization Yes No ("full" curls) -
Tissue homogenization Sonication BeatBox Sonication BeatBox BeatBox
Digestion iST workflow iST workflow
Purification iST workflow (standard) iST workflow (optimized with
WASH 0)
iST workflow (standard)
Table 1 | Overview of compared sample preparation workflows for FFPE tissue (deparaffinized with xylene or “full” curls) and fresh frozen
tissue for LC-MS analysis.
samples were digested and purified according to the FFPE
protocol with the iST 96x Kit (PreOmics GmbH).6 PreOmics’
iST workflow saves time and streamlines the proteomic
sample preparation by employing an easy-to-use, three-step,
all-in-one kit. For samples from FFPE “full” curls without the
xylene-based deparaffinization step, the peptide purification
on the iST cartridge was optimized by adding WASH 0 buffer
to ensure complete removal of paraffin.
LC-MS/MS analysis and data analysis
Peptides were resuspended in LC-LOAD (iST Kit, PreOmics
GmbH), and a 300 ng sample analyzed on an EASY-nLC™
1200 system (Thermo Fisher Scientific) coupled to a timsTOFHT mass spectrometer (Bruker Daltonics) in DIA-PASEF mode
using a 30-minute gradient.
Raw files were analyzed using DIA-NN V1.88 in library-free
mode and searched against the UniProt FASTA database of
Mus musculus (Swiss-Prot entries; downloaded 2022-02-14).
The false discovery rate was set to 1% on the precursor level
and evaluated against decoy precursors. Enzyme specificity
was set as C-terminal to arginine and lysine, using trypsin as
protease, and a maximum of one missed cleavage was allowed
in the database search. Statistical analysis was performed
using Perseus (V 1.6.15.0).
Results and Discussion
The new BeatBox FFPE workflow is faster and more
efficient than traditional sonication workflows
Two major factors influence protocol speed and efficiency:
the deparaffinization method and the parallelization capability
of subsequent sample preparation. Traditional xylene-based
deparaffinization can mean 1.5 hours of additional time with
many manual steps. Furthermore, sample homogenization
may be low throughput and requires repeated time-consuming
cycles if the device is incompatible with multiwell plates.
The BeatBox workflow allows FFPE sample preparation
without xylene-based deparaffinization and can process
up to 96 samples in a plate in parallel. In comparison with
the traditional sonication workflow with xylene-based
deparaffinization, this approach saves up to 4 hours of time
and thereby enables high-throughput processing of 96 FFPE
samples within one working day (Fig. 2).
Figure 2 | Comparison of protocol times between the BeatBox FFPE workflow starting from "full" curl with optimized iST purification using
WASH 0 for paraffin removal and the traditional sonication workflow with initial xylene-based deparaffinization for 96 samples (see also
Table 1).
Traditional sonication workflow
with xylene-based deparaffinization
BeatBox workflow with
"full" curl and Wash 0
Deparaffinization
1h30 2h40 1h
10 min 1h 4h
4h
9h10
5h10 (less than a working day) Parallel homogenization
of 96 samples
De-crosslinking &
protein extraction
iST digestion &
purification incl. Wash 0
iST digestion &
purification
De-crosslinking &
protein extraction
Repeated homogenization 16x10 min cycles for 6 samples each
PreOmics Application Note – FFPE sample preparation simplified with BeatBox: Version 1.0 - For research use only Page | 3 of 5
A xylene-free, high-throughput workflow for in-depth tissue proteome analysis
The BeatBox FFPE workflow permits in-depth proteomic
analyses without prior xylene-based deparaffinization
The BeatBox workflow and the sonication-based workflow
were compared using both “full” curls and deparaffinized FFPE
tissue. Samples were prepared and measured as described in
the materials and methods section (see Table 1 for overview).
For the three different mouse tissue types – cardiac muscle,
kidney, and liver – BeatBox homogenization increased the
proteomic depth on average by 14-43%, depending on tissue
type (Fig. 3). This observation held true for both FFPE tissue
types, deparaffinized using xylene (Fig. 3A) as well as “full”
curls prepared without the xylene-based deparaffinization
step, and only treated with an optimized peptide purification
on the iST cartridge by applying WASH 0 buffer (Fig. 3B).
WASH 0 buffer ensures total removal of the paraffin to prevent
impurities clogging the cartridge or the analytical column.
Figure 3 | Comparison of protein identifications after BeatBox and sonication-based FFPE workflows. FFPE tissue samples from mouse
cardiac muscle, kidney, and liver were homogenized in triplicate either using the BeatBox or a standard sonication device. Samples were
prepared using the iST technology and then analyzed via LC-MS. FFPE samples were either deparaffinized using xylene (A) or used as “full”
curls treated with an optimized iST purification using WASH 0 buffer (B). Error bars represent the standard deviation.
The BeatBox FFPE workflow exhibits remarkable repeatability
Figure 4 illustrates the technical variability of the BeatBox FFPE
workflow. Coefficients of variation (CV) within quadruplicates
from deparaffinized FFPE samples and “full” curl FFPE
samples were below 10% for all assessed tissue types. This
emphasizes the high repeatability of the homogenization
process with BeatBox and makes the workflow ideal for the
proteomic analysis of large sample cohorts.
Using the BeatBox FFPE workflow, both deparaffinized tissue
and “full” curl samples overlapped >91% regarding identified
proteins for all analyzed tissue types (data not shown) and the
number of identified proteins showing valid values differed by
only 1-2% between deparaffinized and “full” curl FFPE tissue
(Figure 3).
The results yield two remarkable findings: First, the resulting
protein IDs reveal a better performance of the BeatBox
compared with the sonication device. Second, the optimized
peptide purification using WASH 0 renders a separate xylenebased deparaffinization step unnecessary, for both the
BeatBox and the sonication-based workflow. Thus, combining
protein extraction on BeatBox with optimized iST-based
digest and peptide purification to process FFPE tissue offers
considerable time savings already at the sample preparation
step and allows for deeper proteomic analyses.
Figure 4 | Assessment of technical variability of the BeatBox FFPE
workflow. Coefficient of variation (CV) in % for protein LFQ intensities
within replicates (n=4, serial sections) from FFPE samples processed
with the BeatBox workflow and analyzed via LC-MS. FFPE samples
were either deparaffinized using xylene (DP) or used as “full” curls
treated with an optimized iST purification with WASH 0 buffer (FC).
The median CVs are shown as numbers next to the violin plots.
Sonication BeatBox
Cardiac
muscle
Kidney Liver
0
3000
2000
1000
4000
5000
6000
7000
A Xylene-based deparaffinization Protein IDs
N = 3
+34%
+21%
+14%
Sonication BeatBox
Cardiac
muscle
Kidney Liver
0
3000
2000
1000
4000
5000
6000
7000
B “Full” curls + WASH 0 Protein IDs
N = 3
+43%
+19%
+28%
%CV for protein LFQ intensities
40
0
180
120
6,9 6,6 6,3 5,9 4,8 5,5
140
160
30
20
10
100
Cardiac muscle Kidney Liver
DP FC DP FC DP FC
PreOmics Application Note – FFPE sample preparation simplified with BeatBox:
A xylene-free, high-throughput workflow for in-depth tissue proteome analysis
Version 1.0 - For research use only Page | 4 of 5
BeatBox workflow processes FFPE and fresh frozen tissue
with similar efficiency
FFPE “full” curls and fresh frozen tissue samples were
prepared with the BeatBox workflow and measured with
LC-MS as described In the materials and methods section
(see Tab. 1 for overview). The resulting data showed that
the number of proteins identified using “full” curls and fresh
frozen samples was comparably high (Fig. 5A), exhibiting an
overlap of 79-87% of shared proteins depending on tissue
type (data not shown). For fresh frozen tissue, roughly 4300,
5800, and 6300 proteins were identified for mouse cardiac
muscle, kidney, and liver tissue, respectively. “Full” curl FFPE
Figure 5 | Comparison of identified proteins and dynamic range obtained from FFPE tissue and fresh frozen tissue homogenized on BeatBox.
FFPE "full" curl samples (FC) and fresh frozen samples (FF) from mouse cardiac muscle, kidney and liver were homogenized in quadruplicate
on the BeatBox, then prepared using iST technology and analyzed via LC-MS. FC samples were treated with an optimized iST purification using
WASH 0 buffer (see Tab. 1 for overview). Shown are proteins identified (A), as well as the dynamic range and protein abundance as S-curves (B).
tissue resulted in only 8-14% lower numbers of proteins
(around 3700, 5200, and 5800, respectively). This shows that
the BeatBox workflow achieves in-depth proteome coverage,
even with FFPE tissue (Fig. 5A).
The dynamic range of protein abundance determined from
LC-MS data of either FFPE “full” curls, or fresh frozen tissue
demonstrated comparable protein depth. This further
confirmed the efficiency of protein extraction from FFPE
tissue when the BeatBox is used (Fig. 5B). This paves the way
to analyze FFPE tissue samples without compromising on the
quality of the results.
Conclusions
Despite being readily available, scientists often refrain
from using FFPE tissue in mass spectrometry (MS)-based
proteomic studies as sample preparation is technically
demanding and often time-consuming. The presented
solution combining the BeatBox and iST technology provides
a simple, fast, and robust way to process FFPE tissue for LCMS-based proteomics. The approach saves up to 4 hours of
time compared with traditional workflows and allows the
preparation of 96 samples in a single working day.
Analyses of FFPE tissue from mouse cardiac muscle, kidney,
and liver showed that the BeatBox workflow yields greater
proteome coverage than a traditional sonication approach.
The results further indicated that optimized washing during
the iST peptide clean-up effectively removed paraffin from
FFPE samples, taking away the need for a separate xylenebased deparaffinization procedure. This workflow is less
toxic, has fewer manual steps, and is highly reproducible,
making it ideal for the analysis of large sample cohorts. Finally,
benchmarking FFPE tissue samples against fresh frozen
tissue showed a similar deep proteome coverage and dynamic
range of protein abundances.
In summary, using the BeatBox workflow to process stored
FFPE tissue from biobanks reduces reliance on fresh frozen
samples and will advance proteomic analyses in a variety of
application areas ranging from basic research to clinically
relevant studies.
“Full” curl + WASH 0 Fresh frozen
Cardiac
muscle
Kidney Liver
0
3000
2000
1000
4000
5000
6000
7000
A Protein IDs
2000 4000 6000
0
2
4
6
8
Log10 intensity
RANK
Liver
2000 4000 6000
0
2
4
6
8
Log10 intensity
RANK
Cardiac muscle
“Full” curl + WASH 0 Fresh frozen “Full” curl + WASH 0 Fresh frozen “Full” curl + WASH 0 Fresh frozen
B
2000 4000 6000
0
2
4
6
8
Log10 intensity
RANK
Kidney
PreOmics GmbH
Martinsried | Germany
PreOmics Inc.
Billerica, MA | USA
Version 1.0 - For research use only Page | 5 of 5
www.preomics.com
References
1. Coscia, F. et al. A Streamlined Mass Spectrometry–Based Proteomics Workflow for Large-scale FFPE Tissue Analysis.
The Journal of Pathology (2020). DOI: 10.1002/path.5420.
2. Obi, NE. et al. Biomarker Analysis of Formalin-Fixed Paraffin-Embedded Clinical Tissues Using Proteomics. Biomolecules
(2023). DOI: 10.3390/biom13010096
3. Piehowski DP. et al. Residual Tissue Repositories as a Resource for Population-Based Cancer Proteomic Studies. Clinical
Proteomics (2018). DOI: 10.1186/s12014-018-9202-4.
4. Kalantari N., Bayani M., and Ghaffari T. Deparaffinization of Formalin-Fixed Paraffin-Embedded Tissue Blocks Using Hot
Water Instead of Xylene. Analytical Biochemistry (2016). DOI: 10.1016/j.ab.2016.05.015
5. Mitsa G. et al. A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized
Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens. International Journal of Molecular Sciences (2022).
DOI: 10.3390/ijms23084443
6. PreOmics protocol: BeatBox Tissue Kit 96x coupled to iST 96x | Formalin-fixed, paraffin-embedded (FFPE) tissue; https://
www.preomics.com/resources
7. PreOmics protocol: iST Kit for FFPE Tissues 8x; https://www.preomics.com/resources
8. Demichev, V., Messner, C.B., Vernardis, S.I. et al. DIA-NN: neural networks and interference correction enable deep
proteome coverage in high throughput (2020). https://doi.org/10.1038/s41592-019-0638-x
Product Manufacturer Product Code
BeatBox Instrument PreOmics GmbH P.O.00144
BeatBox Tissue Kit 96x PreOmics GmbH P.O.00121
iST Kit 96x PreOmics GmbH P.O.00050
WASH 0 (10 mL) PreOmics GmbH P.O.00095
Products Ordering information:
http://www.preomics.com/quote
order@preomics.com
Acknowledgements
We thank Lukas Leiendecker and Anna Obenauf at the Research Institute of Molecular Pathology (IMP) in Vienna, Austria, and
the Histology Facility at Vienna BioCenter Core Facilities (VBCF, member of the Vienna BioCenter), Austria, for providing mouse
tissue samples.
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