Význam tématu
Prostorově řízená multiomická analýza (SpatialOMx) představuje inovativní přístup k mapování molekulárních profilů v tkáních bez potřeby barviv či protilátek. Kombinace MALDI Imaging a LC-MS umožňuje odhalit prostorovou distribuci lipidů, glykanů, metabolitů a peptidů a následně cíleně prozkoumat vybrané oblasti tkáně. Tento postup otevírá nové možnosti ve výzkumu mikroprostředí nádorů, identifikaci molekulárních markerů a zvyšuje přesnost histopatologických diagnostik.
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Innovation with Integrity
TIMS-MALDI MS
MALDI Guided SpatialOMx
timsTOF
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The tumor microenvironment is a
highly variable ecosystem,
giving it an
intrinsic temporal character. De-coding the
cellular communication within the tumor
microenvironment via label-free MALDI
Imaging and x-omics promises to improve the
understanding of the mechanisms responsible
for drug resistance and augment the precision
of histological diagnoses.
Label-free MALDI Imaging monitors
more molecules than antibody or tag-
based imaging techniques.
Map a wide
range of expressed molecules such as
lipids, glycans, metabolites, and peptides
to discover spatially significant expres-
sion. What if you could then deploy a
directed multi-omics approach to decipher
the signaling network at specific sites
within the tumor’s cellular ecosystem?
Use the timsTOF fleX to dive deeper
into the tumor microenvironment
and beyond.
timsTOF fleX combines
the best x-omics platform with a MALDI
source designed for imaging. Intelligence
derived from MALDI Imaging can guide
x-omics analysis of select cell populations
to deliver greater cellular specificity of
LC-MS approaches and establish a new
SpatialOMx benchmark for the future of
pathology.
timsTOF fleX harnesses the power of
SpatialOMx to deliver maximum intelli-
gence per pixel.
MALDI Guided SpatialOMx
Lipids
Metabolomics
Gly
cans
Hist
opatholog
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Intelligent Pixels
0
10,000
20,000
30,000
40,000
50,000
# of P
eptide Sequences
ng on column
25
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40,0
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46,243
31,286
20,836
12,871
1,654
2,414
3,073
3,913
4,562
5,091
Maximize Intelligence per Pixel
An average cell measures approximately 10 μm in diameter. Using MALDI guided laser microdis-
section (LCM) for example, a 50 μm LCM tissue section will contain roughly 25 cells; enough for
bottom-up proteomics analysis on the timsTOF fleX. One instrument that gives you the capability to
do both – high spatial resolution, high speed MALDI and high sensitivity ESI analysis.
Ultimate flexibility and specificity at the click of a software button, comes standard
on the timsTOF fleX.
PEAKS Database Search Results
Data
SpatialOMx Workflow
SpatialOMx is the combination of using MALDI Imaging and ESI to unlock a 5th dimension and show
the distribution of target compounds. On the timsTOF fleX, use the MALDI source to map the distri-
bution of molecules in your sample and identify regions of interest. After extracting and preparing the
sample for LCMS, use the ESI source for the highest level of identifications
3.1
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Why choose Bruker?
timsTOF fleX – The best 4D x-omics and MALDI Imaging system
Since introducing the refleX MALDI-TOF system
in 1992, Bruker has continuously pushed tech-
nological boundaries, developed a wide variety
of applications, and became the uncontested
MALDI market leader. In MALDI Imaging, spectra
are collected spatially, creating a mass spec-
trum at every location which can be projected
as a 2D map.
Single datasets may contain hundreds to thou-
sands of unique, label-free ion images, which can
be used for molecular marker discovery or inves-
tigating molecular content of specific regions.
Bruker has constantly advanced MALDI Imaging
from our patented SmartBeam 3D technology
to SCiLS Lab analysis software. timsTOF fleX
continues this tradition, operating at the industry
standard 20 µm spatial resolution with optional
Zoom Mode down to 5 - 15 µm.
Pioneering the era of
SpatialOMx on the timsTOF fleX
Bruker launches MALDI PharmaPulse, enabling
one million compound libraries to be screened
within a week
FDA approves MALDI Biotyper for clinical
diagnostic use for microbial identification
Bruker launches MALDI Biotyper for microbial ID
Nobel Prize in Chemistry award shared by
Koichi Tanaka of Japan for discovery of MALDI
Richard Caprioli publishes on Tissue lmaging
using MALDI-TOF
Karas, M., Bachmann, D., Bahr, U. &
Hillenkamp, F. Anal. Chem. 57, 2935-2939
( 1985) Matrix-assisted ultraviolet laser
desorption of non volatile compounds time for
analysis to hours
2005
2012
2015
2002
1985
2008
2013
2016
1997
1992
More than 25 Years of MALDI
2018
2019
Bruker launches MRMS instrument scimaX
Bruker launches rapifleX
Bruker launches solariX with dual
MALDI and ESI ion source
Bruker launches Smartbeam-1 laser on
Autoflex and Ultraflex
Bruker launches reflex MALDI-TOF with
world’s first MTP target plate
Bruker introduces reflex, its first commercially
available MALDI-TOF system
2020
First commercialization of
MALDI-2 technology for significantly
enhanced sensitivity and chemical range
Prof. Richard R. Drake, Director, Proteomics Center, Medical University
South Carolina, USA
“ The timsTOF fleX is an innovative instrument that synergizes multiple analytical
capabilities to allow development of novel omics workflows. For imaging MS,
it may be potentially transformative, especially for tissue metabolomics and
glycomic applications.”
Dr. Kristina Schwamborn, Senior Physician, Institute of Pathology,
Technical University Munich, Germany
“ MALDI imaging mass spectrometry goes far beyond microscopy and enables
the assessment of a multitude of analytes in parallel in spatial molecular
arrangements in tissue sections without the need of target specific reagents.
Since the sample remains intact throughout the analysis, it can be stained or
even used for DNA-analysis afterwards. The analysis is fast, has been proven
to be reproducible and no more expensive than other standard pathology tech-
niques like immunohistochemistry. Thus, it has the potential to revolutionize
pathology.”
Dr. Marten Snel, Head of SAHMRI
Mass Spectrometry Core Facility, Australia
“ In my opinion the MALDI enabled timsTOF fleX is a big step forward in this
field. I am confident that timsTOF fleX imaging will have a very positive
impact on our biomedical and clinical research at SAHMRI, especially in
small molecule, lipid and drug imaging”
Trust the Experts
Years of defining the leading edge for technology in MALDI imaging gives Bruker the largest imaging
customer base packed with reference leaders in a wide variety of research fields. Learn what some
of them have to say about how the timsTOF fleX enables SpatialOMx as an essential innovation for
molecular imaging.
Uhlén M et al, 2015. Tissue-based map of the human proteome.
Science. 2015 Jan 23;347(6220):1260419. DOI: 10.1126/science.1260419.
Human Protein Atlas available from www.proteinatlas.org
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Within Tissues
Strict tissue-specific protein expression is uncommon, however, some
types of proteins are predominant - higher molecular weight motor
proteins in muscle, smaller neuropeptides within the brain, digestive
enzymes within the gastrointestinal tract, transport proteins within
the kidney and barrier function-related proteins within the skin. The
proteome of each tissue or organ points to its primary function.
For Regulation of Protein Expression and Celluar Processes
Within any given cell, the processes that regulate life, growth,
functional changes, and death are directed by peptides and proteins.
Transcription factors (generally between 50 - 100 kDa) drive or halt
protein expression from genetic templates. These expressions create
the proteomic means necessary for cell proliferation, differentiation,
or apoptosis, whether in common healthy cellular cycle function
or in response to stress. Similarly, the enzymes that produce post-
translational modifications (e.g., phosphorylation, glycosylation,
methylation) can regulate protein localization, functional activity, and
stability.
For Housekeeping Processes
Housekeeping proteins are expressed at similar levels throughout the
body. “Powerhouse” organelle proteins, such as those in mitochondria
which convert food energy into ATP required by cells, and high molecular
weight scaffolding proteins, such as tubulins and actins, are required
for both maintenance of cellular structure and regular cellular function.
And the Druggable Proteome
Pharmaceuticals target many different types of proteins; greater
knowledge of protein localization, form, and function could improve
drug design and efficacy. Alternative, high(er) affinity binding partners
can more effectively modulate enzymatic activity. Examples include
NSAIDs which reduce pain and inflammation by decreasing production
of prostaglandins by cyclooxygenase (COX) enzymes, or statins which
competitively bind to HMG-CoA reductase to reduce cholesterol.
Commercialized biologics, such as mAbs, often have higher molecular
weight and structural complexity and target specific cell surface
proteins.
Bridging the gap between
4D X-Omics and Pathology
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Brain A highly complex and energy-intensive organ, coordinated
higher functions, e.g. motion, perception, and cognition, are received,
processed and executed in the brain. Neural proteins show specific
expression patterns among cells and structures, as well as in subcellular
structures such as axons, dendrites, and synapses.
Gastrointestinal tissues The gastrointestinal tract (GIT) – the esopha-
gus, stomach, small and large intestines, and rectum – absorbs nutrients
and water, maintains the balance of beneficial microorganisms and
protects against pathogens. GIT proteins are mostly involved in nutrient
breakdown, transport and metabolism, immune response, and tissue
morphology maintenance.
Liver Composed of parenchymal cells (hepatocytes and bile duct cells)
and non-parenchymal cells (sinusoidal endothelial, Kupffer, and hepatic
stellate cells), the liver is the largest internal organ. Liver-specific
proteins include plasma and bile proteins, and proteins associated with
metabolic processes, glycogen storage and detoxification.
Pancreas The pancreas has both exocrine and endocrine functions.
Glandular cells in the exocrine compartment secrete digestive enzymes
into the gastrointestinal tract, while the islets of Langerhans execute the
pancreatic function, secreting insulin and other hormones. Many
pancreatic mRNAs encode specialized secreted proteins.
Skin The skin (epidermis, dermis and subcutaneous layer) is a sensory
organ and a protective barrier. The epidermis is mostly keratinocytes
which protect against physical, chemical and biological insults. Most
protein functions are related to squamous cell differentiation and cornifi-
cation, pigmentation, and hair development.
Kidney Primary functions of the kidney include maintaining body
homeostasis by regulating blood composition and eliminating waste.
Different cell types are organized into sub-anatomical structures with
distinct functions, showing elevated levels of essential proteins, e.g.
proteins required for blood filtration are elevated in the glomerulus.
Lung The lungs are primarily responsible for respiration: the gaseous
exchange of O
2 and CO2 between air and blood occurs in ~300 million
alveoli. Pneumocytes, bronchial epithelium, and the endothelial cells
facilitate O
2/CO2 exchange, while alveolar macrophages protect against
potential infection from inhaled microbes.
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Sample preparation
Data acquisition
1. Cryosections of mouse
stomachs (WT, NT, T, TS)
2. Sample lysis
3. Digestion
Column:
C18 (25 cm x 75 µm, 1.6 µm,
IonOpticks, Australia)
Flow rate:
400 nL/min
Oven temp.:
50°C
Source:
CaptiveSpray
Acquisition method:
PASEF
Mass Range:
100 – 1700 m/z
Total cycle time:
1.1 s
Number of MS/MS ramps: 10 PASEF scan at 100 ms
(each MS/MS PASEF
scan contains 12 MS/MS)
Time [min] %B
0
5
90
35
110
95
120
95
121
5
130
5
LC separation
MS detection
Biological replicates
Sa
mple pr
eparation
re
plic
ates
The timsTOF fleX offers a combination of two
unique technologies, Trapped Ion Mobility
Spectrometry (TIMS) to enhance ion separation
and Parallel Accumulation Serial Fragmentation
(PASEF) to improve ion utilization efficiency and
data acquisition speed. The performance of the
timsTOF fleX mass spectrometer with PASEF for
label-free quantitation of proteins can be demon-
strated on proteins extracted from sectioned
mouse tissue. In brief, more than 5000 protein
groups could be reliably identified and quantified
from 240 ng protein per sample using 90-minute
gradients. The optimized PASEF method used
on the timsTOF fleX gave very high technical
reproducibility which is an important prerequisite
for label-free quantitation (LFQ).
Furthermore, the complete process (including
tissue preparation, digestion, and data acquisi-
tion) was highly reproducible, which is critical in
the application of proteomics to clinically relevant
specimens. When comparing the proteome com-
position of tumor and non-tumor tissue, gene
ontology analysis indicated the enrichment of the
minichromosome maintenance protein com-
plex (MCM-complex) in tumor over non-tumor
samples. The MCM-complex has been shown
as an essential component of the pre-replica-
tion complex (pre-RCs), which is involved in
DNA replication initiation and the recruitment of
DNA-Polymerases. In various studies a malfunc-
tion of the MCM-complex has been linked to
genomic instability, increased cell proliferation,
and a variety of carcinomas.
4D X-Omics Workflows −
Just a Click Away
Label-free quantitation on the timsTOF fleX with PASEF: investigation of
proteomic changes in tissue samples of mouse gastric carcinoma.
Workflow for the analysis of isolated mouse stomach tissue using label-free quantitation
Novel mass spectrometry imaging workflow: Automated annotation of metabolites and lipids from tissue
*Lipid Maps and HMDB are not Bruker products.
SpatialOMx Automated Molecular
Annotation Workflow
SCiLS Lab – Industry Leading Imaging Software
For SpatialOMx a new mass spectrometry imaging workfl ow with automatic metabolite annotation
will be supported by SCiLS Lab and MetaboScape: regions of interest can be transferred from SCiLS
Lab to MetaboScape and the annotated peak lists can be loaded into SCiLS Lab for visualization of
spatial compound distribution.
• Vendor-neutral analysis and visualization • Quantify target molecules directly from tissue
• New SpatialOMx workfl ow for automatic metabolite annotation
timsTOF fleX data
Bruker's MALDI Imaging solutions
consists of established sample preparation
protocols, covers fully integrated hardware
and software control, all the way to analysis
workflows.
Discover regionally specific molecular markers
and biochemical changes
Wide range of applications including
proteomics, lipidomics, glycomics,
inorganic compounds and clinical research
Localize and quantify for drug discovery
Visually explore metabolic pathways
Correlate molecular changes to disease
Acquire MALDI
Imaging Data
Import spectral and ROI
data into MetaboScape to
annotate compounds
Review and export
compound annotations to
SCiLS Lab
MSI peak
list data
ROI
information
Set up projects
in SCiLS Lab and
define ROIs
Review images in
SCiLS Lab with
annotated compounds
Novel Mass Spectrometry Imaging Workflow
Compound
annotations
While many researchers can utilize spatialOMx “out of
the box”, customers with challenging workflows asked
for more. Research centered around small molecules
and lipids typically test the limits of MALDI sensitivity
and molecular coverage.
The answer is MALDI-2.
Bringing Enhanced
Depth and Sensitivity
The SpatialOMx enabled timsTOF fleX
represents an entirely unique solution
for adding biological context to routine
OMICS or pharma studies.
MALDI-2 enables access to chemical
classes typically prone to ion suppres-
sion in MALDI
Sensitivity boost by up to 2-3 orders
of magnitude compared to MALDI,
depending on sample, matrix and
analyte
No physical hardware changes needed,
switch between MALDI and MALDI-2
by one click in the software
User friendly software solution, easy
instrument calibration and application
scientist tested methods to start mea-
surements immediately
Pharma - Move beyond toxi-
cology to PK/PD and more with
the ability create images from
tissue at previously unreachable
sensitivity.
Metabolites - Image metabolic
classes and pathways previously
undetectable by MALDI alone.
Prof. Klaus Dreisewerd,
Leader Section Biomedical
Mass Spectrometry,
University of Muenster, Germany
“In the last 35 years, MALDI has
become a unique and rapid analytical tool for
a wide variety of applications. We developed
MALDI-2 to significantly extend the technique
by providing much higher sensitivity for small
molecules and inclusion of chemical classes that
didn’t traditionally ionize. With an extensive set
of unique features, the MALDI-2 empowered
timsTOF fleX will take MALDI to new frontiers
previously not available.“
MALDI
MALDI-2
m/z 369.35, [Cholesterol-H
2O+H]
+
m/z 792.55, [PE(40:6)+H]+
m/z 810.68, [GalCer(d18:1/C24:0)+H]+
1
2
Originally developed by the Klaus
Dreisewerd group at the University of
Muenster, MALDI-2 uses laser post-ion-
ization to enhance and enrich the MALDI
experiment providing access to chemical
classes typically opaque to MALDI with
sensitivities never seen before on any
platform*.
Post-ionization leads to a significant
boost in ion yields and a reduction of ion
suppression effects, resulting in increas-
ingly complex spectra. In this context,
de-convoluted feature assignment in the
TIMS mobilogram becomes increasingly
useful. Next to finding a larger number
of features, they are also described by
not one but two independent measures,
enabling confident identification.
* 1. Soltwisch, J. et al. Mass spectrometry imaging with laser-induced postionization, Science, 2015, 348, 211-215.
2. Barré, F. P. Y. et al. Enhanced Sensitivity Using MALDI Imaging Coupled with Laser Postionization (MALDI-2)
for Pharmaceutical Research, Anal. Chem., 2019, 91, 10840-10848.
MALDI
MALDI-2
20,000
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m/z
15,000
10,000
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-15,000
-20,000
5,000
-5,000
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int
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MALDI
m/z
1.25
1.30
1.35
1.40
1.45
m/z
1/K
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m/z 852.54
m/z 852.54
1/K
0 1.43
and 1.48
1/K
0 1.43
Step 1: Laser hits the sample surface and
desorbs material. Some ions and neutral
molecules are generated.
Step 2: A second laser intercepts the
evolving plume and postionizes neutral
molecules, which enhances the ion yield.
MALDI-2
MALDI Guided SpatialOMx
timsTOF
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Bruker Daltonik GmbH
Bremen · Germany
Phone +49 (0)421-2205-0
Bruker Scientifi c LLC
Billerica, MA · USA
Phone +1 (978) 663-3660
[email protected] - www.bruker.com
For research use only. Not for use in clinical diagnostic procedures.
Scan the QR-Code
for more Details
timsTOF fleX uniquely
enables SpatialOMx
PASEF powered LC-MS/MS identification matched
with spatial localization identifies and locates multilevel
genomic expression in tissue without labels
timsTOF fleX provides results
without compromise
All the 4D-Omics power that you demand from proven
PASEF workflows with fast, software-controlled
changeover to MALDI for rapid molecular imaging
timsTOF fleX allows
you to work smarter
Label-free mapping of metabolites, lipids, glycans,
peptides and more can efficiently direct your deep
4D-Omics studies with laser guided precision so that
you focus on the tissue regions that matter.
