Bruker timsTOF fleX - MALDI Guided SpatialOMx

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

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Intelligent Pixels

0

10,000

20,000

30,000

40,000

50,000

# of P

eptide Sequences

ng on column

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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

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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

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2012

2015

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2016

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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

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LC separation

MS detection

Biological replicates

Sa

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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

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MALDI

m/z

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m/z 852.54 

m/z 852.54

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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.

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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.