The authors would like to acknowledge the Sydney Medical School Foundation and Blues and Foundation for funding part of this work through their PhD scholarship program for Alzheimer's Disease Research and an ARC Discovery grant (DP160102063) awarded to PKW.

CAUTION: All applicable safety precautions should be followed when performing this procedure, including the use of appropriate personal protective equipment (PPE) (e.g., lab coats, nitrile gloves, safety glasses, etc.)
1. Preparation of Reagents and Equipment
<ol>
	<li>Preparation of solutions
	<ol>
		<li>To prepare 500 mL of Carnoy&#39;s fluid, mix 100% ethanol (EtOH), chloroform, and glacial acetic acid in a 6:3:1 v/v/v ratio in a clean glass Schott bottle.</li>
		<li>To make liquid nitrocellulose (NC), dissolve NC membrane (commonly used in western blotting) in neat acetone, by gentle agitation, to a final concentration of 40 mg/mL.</li>
	</ol>
	</li>
	<li>Preparation of NC coated slides
	<ol>
		<li>Use a technique similar to the one used for the preparation of blood smears for histological examination20 to prepare NC coated slides.</li>
		<li>Pipette 40 &#181;L of liquid NC onto one edge of a conductive indium tin oxide (ITO) microscope slide. Using a regular glass microscope slide, drag the NC under surface tension across the slide to create an even thin film coating.</li>
		<li>Allow the NC to dry at room temperature for 20 s and then store until needed at room temperature. Storage of samples under these conditions can be indefinite provided the tissues are maintained and free of dust. 
		NOTE: This method of preparation is better known as &#34;thin film&#34; and will produce a uniform coating that relies on the viscosity of the NC as well as its lack of surface tension to self-level on the slide. The only care that should be taken when preparing the slides is not moving too quickly, which will create streaks of NC rather than a complete coverage. 
		&#8203;CAUTION: As liquid NC dries very quickly, it is essential to work quickly to avoid drying the solution before it has been properly spread. Care should also be taken when handling NC in its liquid state, as it is a highly energetic compound and should not contact a source of ignition to prevent incendiary explosion. NC also undergoes endothermic transitions when drying and can cause cold burns if allowed to come into prolonged contact with skin or gloved hands. To minimize all associated risks, only small amounts should be prepared at each time (&#60;1 mL is recommended). Once dry it is stable at room temperature. However, NC can remain explosive when present in large quantities.</li>
	</ol>
	</li>
	<li>Creation of custom vapor chambers
	<ol>
		<li>Cut a piece of thick blotting paper with a pair of scissors big enough to fit the bottom half of a standard plastic petri dish (94 mm in diameter and 3 mm thick).</li>
		<li>Cut the paper, leaving a rectangular strip in the center, the same size as a microscope slide, leaving enough excess so that the paper will maintain its position in the Petri dish (Supplementary Figure 1).</li>
	</ol>
	</li>
</ol>
2. Preparation of Tissue Sections
<ol>
	<li>FFPE Tissue21.
	<ol>
		<li>Select the desired tissue block and section it at 12 &#181;m thickness in a standard bench mounted microtome and float mount onto the NC pre-coated ITO slides.</li>
		<li>Submerge the slides in fresh xylene to remove residual paraffin for 2 min, and repeat the operation a second time. 
		NOTE: If the paraffin block is especially pervasive or the tissue is relatively small compared to the paraffin block, the samples can be heated at 60 &#176;C to melt the majority away prior to washing in xylene.</li>
		<li>Wash the deparaffinized samples by submerging the slides in a graded solvent series: 70% v/v EtOH/water, 100% EtOH, Carnoy&#39;s fluid, 100% EtOH, ultra-pure water and 100% EtOH. The duration of each submersion should be 30 s, except for Carnoy&#39;s fluid, which must last 2 min. 
		&#8203;CAUTION: Carnoy&#39;s fluid and xylene must be stored and used in a fume hood, as they are highly toxic if inhaled.</li>
	</ol>
	</li>
	<li>Formalin-Fixed Frozen (FFF) Tissue 
	NOTE: Samples should already be frozen, appropriately according to the sample type.
	<ol>
		<li>Equilibrate the tissue block at the operating temperature of the microtome for 20 min22. 
		NOTE: Equilibration temperature is dependent on the type of tissue.</li>
		<li>Section the tissue using a microtome at 12 &#181;m and thaw mount the sections onto a pre-prepared NC coated ITO slide.</li>
		<li>Store the slides in a vacuum desiccator on the bench in the dark. 
		NOTE: Samples can be stored for several weeks under these conditions.</li>
		<li>Wash the samples in a graded solvent series as stated for FFPE tissue (2.1.3). 
		NOTE: There is no need for a xylene deparaffinisation step as the sample is not embedded.</li>
	</ol>
	</li>
</ol>
3. Methylene Crosslink Hydrolysis
<ol>
	<li>Load the sample slides (either FFF or FFPE) into a plastic slide box which is filled to the top with 20 mmol tris-HCl (pH 8.8). Seal the box and place it in a water bath, containing 500 mL of water, allowing the box to touch the bottom. Then heat it for 15 min at 120 &#176;C in a pressure cooker which can achieve an operating pressure of 70 kPa.</li>
	<li>Remove the slides, allow them to cool, and let them dry at ambient temperature for 15 min.</li>
</ol>
4. Tissue Digestion
<ol>
	<li>Once hydrolyzed, coat the samples with 10 &#181;L of trypsin solution (1 mg/mL) in ultra-pure water, by pipetting 10 &#181;L of the solution onto the edge of the tissue section then, using the same pipette tip, drag the droplet across the whole surface of the tissue under surface tension. 
	NOTE: Avoid scratching the tissue surface with the pipette tip and over spreading the enzyme beyond the borders of the tissue edges.</li>
	<li>Allow the samples to dry at ambient temperature.</li>
	<li>Once dry, mount the samples inside the top of the previously constructed vapor chamber (tissue facing down) with autoclave tape on either edge of the slide (Supplementary Figure 2).</li>
	<li>Pipette 600 &#181;L of a solution containing a 1:1 v/v mix of 100% acetonitrile and 50 mM ammonium bicarbonate carefully onto the blotting paper finger in the bottom part of the petri dish until the finger appears to be evenly wet.</li>
	<li>Place the top half of the vapor chamber on the bottom half, ensuring the paper finger and sample slide align perfectly, and seal the chamber around its equator with paraffin film.</li>
	<li>Leave the sample overnight in a 37 &#176;C incubator to allow complete digestion.</li>
</ol>
5. Matrix Coating via Sublimation
<ol>
	<li>Once digested, weigh the sample slide on a 5-digit microanalytical balance.</li>
	<li>Mount the slide onto the cooling finger of the sublimation apparatus and secure it with copper tape, in the same way as described for the vapor chamber (Supplementary Figure 3). 
	NOTE: To ensure that the slide is contacted evenly by the cooling finger, a layer of copper tape is placed on the bottom of the cooling finger to level the surface. This ensures that the slide is evenly cooled which leads to even deposition of matrix.</li>
	<li>Place 300 mg of &#945;-cyano-4-hydroxycinnamic acid (CHCA) matrix into a glass petri dish at the bottom of the chamber and spread evenly to create a thin layer of matrix crystals.</li>
	<li>Assemble the sublimator and secure the two halves with the horseshoe clamp. Suspend the assembled unit 15-20 cm above a sand bath, preheated at 220 &#176;C, by placing it in a metal ring connected to a retort stand.</li>
	<li>Connect the chamber to the vacuum source, engage the vacuum, and allow it to stabilize down to ~25 mTorr for 5 min.</li>
	<li>Pack the cooling finger to the top with ice and add 50 mL of water. Allow the apparatus to settle for a further 5 min before proceeding.</li>
	<li>Lower the chamber onto the surface of the sand, ensuring the sand completely contacts the bottom of the chamber, and leave it for 45 min to create an ideal coating of 0.22 mg/cm2 
	CAUTION: Care should be taken when handling glassware at high vacuum, as any damage to the apparatus, while evacuated, can result in catastrophic failure of the glass chamber.</li>
	<li>After 45 min, remove the chamber from the sand bath by raising the metal ring and vent the chamber. 
	NOTE: Once the chamber has been vented, the slide must be removed very quickly as water in the air will begin to condense on the freezing finger and sample slide.</li>
	<li>Remove the slide and weigh it to ensure the desired coating has been achieved. 
	NOTE: If insufficient coating has been achieved, simply repeat the sublimation process for the slide until the desired coating thickness has been achieved.</li>
</ol>
6. Recrystallization
<ol>
	<li>Once sublimated, mount the sample slide inside the top of the previously constructed vapor chamber, as described earlier (tissue facing down).</li>
	<li>Pipette 600 &#181;L of a solution containing a 1:1 (v/v) mix of 100% acetonitrile and 0.1% v/v trifluoroacetic acid in water carefully onto the blotting paper in the bottom part of the petri dish to ensure an even coating.</li>
	<li>Assemble the chamber, ensuring the paper tab and microscope slide align perfectly, and leave in a 37 &#176;C incubator for 1 h. 
	Caution: DO NOT seal the chamber. 
	NOTE: Once recrystallized, the normally yellow hue of the matrix should change to white, look less shiny, and appear very even. This indicates that recrystallization has been performed correctly.</li>
	<li>The sample is now ready for analysis.</li>
</ol>
7. Instrumentation
<ol>
	<li>Scan the slide in a flatbed scanner to create a digital image.</li>
	<li>Load the sample into the mass spectrometer and then analyze using the appropriate software platform.</li>
	<li>Analyze the samples in positive ion reflectron mode with a mass range of 750-3,500 Da, with a spot resolution that is applicable to the desired result. In most cases, 20-50 &#181;m raster width is sufficient, however as little as 10 &#181;m can be used15. 
	CAUTION: MALDI UV lasers are a source of ionizing radiation and should not be viewed directly. Ensure that the laser is contained within instrument shielding prior to operation.</li>
</ol>

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The authors have no conflict of interest or commercial interest to disclose

This protocol was designed to maximize the generation of ionizable molecular species while eliminating delocalization of analytes. The key factors involve using the same overriding principle when applying matrix, digesting the sample, or recrystallizing after sublimation24; namely, that an even deposition of vapor, matrix or otherwise, needs to be created and maintained. Pipetting solvent washes for recrystallization and digestion, evenly underneath the sample, prevents any individual area receivi...

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has been employed as an image based technique for two decades1,2, analyzing a range of biomolecules including: lipids3, peptides2,4, proteins2,5, metabolites6,7, N-glycans8, and synthetic molecules such as therapeutic drugs9,10. The number of publications demonstrating the utility of this technique have grown significantly over the last decade6,11,12,13. Certain molecules, such as lipids, are relatively easy to analyze via MALDI MSI, as they ionize readily due to their chemical nature and thus require little prior preparation3. However, for more difficult targets such as peptides, the steps required to effectively ionize these molecules are extensive and generally complicated14. There are currently very few publications that aim to address or demonstrate reproducibility in the methodologies that are employed to prepare tissue for this unique visual technique15. For this reason, we have compiled observations and implemented optimizations into a single, easy to implement, methodology that should require little to no modification, for the analysis of peptides from a formaldehyde cross-linked tissue source14.
In this manuscript, we have described a validated, low cost reproducible methodology for the detection and spatial mapping of peptides, generated from formalin-fixed frozen (FFF) and formalin-fixed paraffin-embedded (FFPE) tissue sections. This methodology does not require or rely on any specialized instrumentation3. Specifically, we address the many aspects of specialized sample preparation necessary to analyze peptides; steps such as antigen retrieval16 and matrix coating. Our protocol also utilizes inexpensive equipment and reagents, thereby making this methodology accessible to a wider community who would otherwise be unable to afford the alternate robotic apparatus17.
The reasoning behind developing a manual sample preparation method was two-fold: Firstly, the use of a sublimator creates a consistent and homogenous coating of matrix crystals that are ~1 &#181;m in length18, something unachievable with more common spraying techniques. Secondly, the relatively small set up costs: the total cost of the custom apparatus was &#60;$1500 AUD. We note, in terms of cost effectiveness, the price per sample is far cheaper when there is no robotic machinery involved. The use of sublimation has been reported previously, however, to the best of our knowledge, step-by-step methodologies that describe this process and sample preparation have not been reported nor described in the literature.
This protocol is intended to assist researchers that have access to a MALDI mass spectrometer and who are intent on generating spatial information in relation to a bio-molecule of interest19. In essence, MALDI MSI is a form of histological screening that does not rely on antibodies or stains2.

<table><tbody><tr><td>Cryo Microtome</td><td>Leica</td><td>CM3050</td><td>For preparation and section of tissue.</td></tr><tr><td>Indium Tin Oxide Microscope slides</td><td>Bruker</td><td>8237001</td><td>For preparation and section of tissue.</td></tr><tr><td>Coplin Jars</td><td>Sigma Aldrich</td><td>S5516</td><td>For preparation and section of tissue.</td></tr><tr><td>Pressure Cooker</td><td>Kambrook</td><td>KPR620BSS</td><td>For preparation and section of tissue.</td></tr><tr><td>Sublimator</td><td>Chem Glass</td><td>NA</td><td>For sublimation procedure. Similar in design to the CG-3038 however it was custom made&amp;nbsp;</td></tr><tr><td>Sand bath</td><td>NA</td><td>NA</td><td>For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company</td></tr><tr><td>Glass Petri Dish</td><td>Sigma Aldrich</td><td>CLS70165100</td><td>For sublimation procedure.</td></tr><tr><td>Vacuum Pump</td><td>NA</td><td>NA</td><td>For sublimation procedure. Sourced as a spare part from an old mass spectrometer&amp;nbsp;</td></tr><tr><td>Cold trap</td><td>Chem Glass</td><td>CG-4510-02</td><td>For sublimation procedure.</td></tr><tr><td>Hot Plate</td><td>John Morris</td><td>EW-15956-32.&amp;nbsp;</td><td>For sublimation procedure.</td></tr><tr><td>Plastic petri dish</td><td>Sigma Aldrich</td><td>Z717223</td><td>For sublimation procedure.</td></tr><tr><td>37 &amp;deg;C incubator</td><td>NA</td><td>NA</td><td>For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old&amp;nbsp;</td></tr><tr><td>Blotting paper</td><td>Sigma Aldrich</td><td>P7796</td><td>For sublimation procedure.</td></tr><tr><td>Nitrocellulose&amp;nbsp;</td><td>Sigma Aldrich</td><td>N8395</td><td>For washing of slides.</td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>650501</td><td>For washing of slides.</td></tr><tr><td>Xylene</td><td>Sigma Aldrich</td><td>214736</td><td>For washing of slides.</td></tr><tr><td>100% EtOH</td><td>Sigma Aldrich</td><td>1.02428</td><td>For washing of slides.</td></tr><tr><td>70% EtOH</td><td>Sigma Aldrich</td><td>NA</td><td>For washing of slides. Made in lab from 95% stock ethanol&amp;nbsp;</td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>C2432</td><td>For washing of slides.</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma Aldrich</td><td>ARK2183</td><td>For washing of slides.</td></tr><tr><td>Tris HCL pH 8.8</td><td>Sigma Aldrich</td><td>TRIS-RO</td><td>For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8</td></tr><tr><td>Milli Q Ultra-Pure Water</td><td>Sigma Aldrich</td><td>NA</td><td>For proteolytic cleavage. Purification performed in house by sartorious water purification system</td></tr><tr><td>Ammonium Bircarbonate</td><td>Sigma Aldrich</td><td>A6141</td><td>For proteolytic cleavage.&amp;nbsp;</td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>T0303</td><td>For proteolytic cleavage.&amp;nbsp;</td></tr><tr><td>CHCA Matrix</td><td>Sigma Aldrich</td><td>C2020</td><td>For recrystallisation.</td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>1.00029</td><td>For recrystallisation.</td></tr><tr><td>Trifluoroacetic Acid (TFA)&amp;nbsp;</td><td>Sigma Aldrich</td><td>302031</td><td>For recrystallisation.</td></tr></tbody></table>

optimal preparation of formalin fixed samples for peptide based matrix assisted laser desorption/ionization mass spectrometry imaging workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

If followed correctly, this protocol produces images that clearly represent the gross morphology of the tissue without any scratches or other deformations (Figure 1). The ideal validation for a correctly performed sample preparation, is the ability to distinguish between different physical structures by changing the molecule being viewed (Figure 2).
A good guide for det...

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Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

optimal-preparation-formalin-fixed-samples-for-peptide-based-matrix

Research

JoVE Journal

Biochemistry

8.8K Views.  University of Sydney. Dit protocol beschrijft een reproduceerbare en betrouwbare methode voor de sublimatie gebaseerde voorbereiding van formaline vaste weefsels die bestemd zijn voor imaging Spectrometrie van de massa.

Video: Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Medicine

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Bioengineering

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Immunology and Infection

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Biology

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

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