TITLE: A selective process for application of EDC and 4-APEBA for carbonyl containing metabolites by MALDI-MSI
AUTHORS: Kevin J Zemaitis, Christopher R Anderton, Dusan Velickovic

[DESCRIPTION]
Herein, we outline the protocol for application of 4-(2-((4-bromophenethyl)dimethylammonium)ethoxy)benzenaminium dibromide (4-APEBA) which we have previously demonstrated to be an effect reagent in tandem with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) to target carbonyl containing metabolites in-situ for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Either with or without the selective application of EDC, MALDI-MSI is able to differentiate ketones and aldehydes from carboxylic acids lending more confidence to annotations. This has enabled metabolic pathway analyses with high-levels of coverage routinely enabling the detect of over a hundred metabolites containing carbonyls. 

This reagent was previous introduced for LC-MS/MS purposes (see references), but generally through our on-tissue/on-target chemical derivatization (OTCD) protocol we find that (1) we have increased sensitivity and coverage of primary metabolites, (2) we have increased confidence within our annotations due to bromine isotopic patterns, (3) we do not delocalize metabolites within the process of OTCD, (4) we have multiplexed detection of non-derivatized metabolites and lipids in tandem with OTCD products, and (5) this is applicable to a broad variety of mammalian or plant tissues, and microbial cultures.

[STEPS]
SECTION: Materials for OTCD
3. All chemicals were used as received without any further purification, and synthesis of 4-APEBA was completed in house:

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride - CAS Number: 25952-53-8 - Sigma-Aldrich (>98.0%; St. Louis, MO) or Tokyo Chemical Industry Co. (>98.0%; Portland, OR)
4-(2-((4-bromophenethyl)dimethylammonium)ethoxy)benzenaminium dibromide (4-APEBA) - CAS Number: 1226984-28-6 was synthesized at PNNL according to protocols outlined elsewhere (Note: several chemical suppliers do readily stock this reagent) 
2,5-dihydroxybenzoic acid (DHB) - CAS Number: 490-79-9 - Sigma-Aldrich (98%; St. Louis, MO)
Methanol (MeOH) - Fisher Chemical (HPLC grade; Fair Lawn, NJ)
Water (H2O) - Fisher Chemical (HPLC grade; Fair Lawn, NJ)
SECTION: Instrumentation for OTCD
2. 1. M5-Sprayer (HTX Technologies, Chapel Hill, NC) or equivalent
Related HTX Sprayers also can be used (this was tested on a TM-Sprayer as well).

2. AZURA Pump P4.1S (Knauer GmbH, Berlin, Germany) or equivalent
We utilize this pump with a 5 mL loop equipped for matrix deposition, usually within this process DHB is loaded and once EDC/4-APEBA is deposited lines can be cleaned the switched, and the process is for the third step of matrix deposition.
The loop size can be smaller or the use of a secondary pump is not necessary if an external syringe pump is equipped.

3. External syringe pump
We utilize the syringe pump for EDC/4-APEBA deposition due to low volumes of reagent we use initially to keep cost of OTCD at a minimum with a large loop on our Knauer pump.
We use a 1 mL Hamilton gastight syringe (Model 1001 TLL, PTFE Luer Lock) connected to the secondary swage passthrough on the M5 Sprayer.
SECTION: Considerations Prior to Starting Work
1. The points below are distilled from 100's of applications on various samples, and commonalities found to streamline the on-tissue/on-target chemical derivatization (OTCD) process and minimize batch effects. From experience, we suggest considering the following prior to starting work: 
SECTION: EDC and 4-APEBA Application
4. Both EDC and 4-APEBA are diluted within H2O after the sample is mounted. No old reagents are kept or used within our process, and all reagents and matrix are applied using the HTX sprayer (reference below).


 

SECTION: DHB Application
5. Directly following the OTCD steps (either solely 4-APEBA or EDC/4-APEBA), matrix is applied. This deposition was previously optimized within the reference below. While DHB does contain moieties that can be derivatized, we found any derivatized DHB did not effect sensitivity. Additionally, these products provide additional lock masses that can be exploited for MS calibration.
 

SECTION: MALDI-MSI Analyses
6. A variety of MALDI instruments have been used for the collection of 4-APEBA OTCD datasets.

Both a 7T scimaX FTICR MS and 12T solariX XR FTICR MS (Bruker Daltonics, Bremen, DE), as well as a timsTOF fleX MS (Bruker Daltonics).

Using at least one lock mass is recommended, ideally the [4-APEBA-H20]+ corresponding to C18H24N2OBr at m/z 363.1067, alternatively additional masses of [DHB+4-APEBA]+ or other reaction byproducts and matrix clusters can be employed as well.
SECTION: EDC and 4-APEBA Application
4.1. The following method outlined within Table 1 is used for both deposition of EDC and 4-AEPBA, separately.

 


SECTION: EDC and 4-APEBA Application
4.3. Note: 4-APEBA OTCD can be exploited for determining specific moieties on molecules (e.g., ketones/aldehydes vs caboxylic acids) as shown within Veličković et al. 2024 referenced below.

If EDC is not applied, caboxylates will not form an O-acylisourea intermediate, resulting in derivatization
of only ketones and aldehydes
If EDC and 4-APEBA are applied in sequentially, derivatization will occur for ketones, aldehydes, and carboxylic acids
 
If following this protocol, it is highly recommended to complete EDC/4-APEBA and sole 4-APEBA deposition for the comparison.
SECTION: EDC and 4-APEBA Application
4.2. After final deposition of 4-APEBA, an aqueous and organic wash of several syringe volumes are used to clean the HTX Sprayer lines and head according to laboratories best practice.
SECTION: Considerations Prior to Starting Work
1.1. After initial methods validation, for completing small or large cohorts portion, weigh all derivatization (EDC/4-APEBA) reagents in 1-2 mL autosampler vials. Store under proper conditions per manufacturer guidelines within the freezer (-20°C) or fridge (2-4°C) for the duration of the project. 

While some manufacturers mention reagents can be stored as solutions, portion enough solid to reconstitute as 1-2 mL of solution within kits for each project.
Dispose of all excess reagents after primary use.
SECTION: Considerations Prior to Starting Work
1.2. Ensure all materials are present and instruments are working properly when you start the OTCD process. 

This OTCD reaction is a quick process, where incubation times beyond the time it takes to spray the reagents is not needed (nor is rehydrating the slides). 
It is best practice to be cognizant of timings, where induction of additional dry incubation periods before analyses can induce batch effects.
Derivatized metabolites were found to be stable for several days. 
SECTION: Considerations Prior to Starting Work
1.3. Always drop-cast metabolite standards of interest onto the slides near the tissue/target sample (e.g., 1 μL of ~mM ketone, aldehyde, or carboxylic acid) for QA/QC. 

Make stock standard solutions and aliquot into vials for all samples within the analyzed cohort, ensure enough water of solutions is sufficient enough for adequate surface tension (e.g., ~50% MeOH).
Mark all positions of dried droplets, and image the entire droplet. This ensures that chemical reactions were not problematic, and enables QA/QC metrics to be determined based on derivative standards intensities.
When comparing in-tissue annotations it is best to exclude these ROIs, as it will effect maximum intensity color scales.
SECTION: Considerations Prior to Starting Work
1.4. When switching between EDC and 4-APEBA, properly rinse the sprayer line and the syringe.

Maintaining EDC/4-APEBA deposited ratios will ensure that applied matrix will be able to co-crystalize efficiently and there will not be larger matrix effects from OTCD (of the matrix itself). It is not recommended to combine EDC/4-APEBA into one single deposition, deposit individually in a two step process.
SECTION: DHB Application
5.1. The following method outlined within Table 2 is used for both deposition of DHB.

 
. 
SECTION: DHB Application
5.2. After DHB matrix application, MALDI-MSI analyses can be performed.
SECTION: Data Analyses
7. 4-APEBA-based OTCD MSI data is annotatable using METASPACE (https://metaspace2020.eu/) with the chemical modifier of (+C18H22N2Br, +345.0966 Da).

Filtering using the adducts tool searching for [M]+ adducts with the chemical modifier (+C18H22N2Br) and starting with FDR of 10% is a good starting point.

It is vital to use the diagnostics tool, and also search through the full profile data in a different visualization package to ensure proper annotation of Br isotopic pattern.

You may find at >20% FDR there can be annotations which are true positives, but were annotated with low FDR due to isotopic complexity.

Additionally, [M+H]+ or [M+Na]+ or [M+K]+ of biogenic amines and other primary metabolites or phospholipids are commonly detected as well. Note, the carboxyl ester of the acyl chains within lipids are protected functional groups that are not able to be derivatized.