Trifluoroacetic acid tfa

Cytochrome c from equine heart (ref. C7752: MW~12.4 kDa, purity ≥ 95%), acetic acid (glacial), and Dulbecco’s Phosphate Buffered Saline (pH 7.4) were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium chloride and sodium acetate were obtained from Fisher Scientific (Loughborough, UK) and Fluka (Buchs, Switzerland), respectively.
Silver wire and silver chloride (AgCl) for the preparation of electrodes were obtained from Sigma Aldrich (Steinheim, Germany). PVC tubing (3 mm ID, 5 mm OD, and 1 mm wall) and agarose (used to prepare salt bridge assemblies) were obtained from VWR International AG (Dietikon, Switzerland) and Conda (Madrid, Spain), respectively.
The organic solvents, acetonitrile (ACN) and methanol (MeOH), were HPLC grade (Fischer Scientific, Loughborough, UK). Trifluoroacetic acid (TFA; 99% extra pure) was obtained from Acros organics (Geel, Belgium). Ultra-pure water (Millipore Milli-Q Gard 1 Purification pack resistivity > 18 MΩ.cm; Zug, Switzerland) was used to prepare all solutions. All other chemicals were at least of analytical grade.

Chemicals: Methanol (Optima LC/MS grade) and acetonitrile (Optima LC/MS grade) were purchased from Fisher (Illkirch, France), formic acid from Sigma Aldrich (St Quentin Fallavier, France). Ultra-pure water was obtained using a Milli-Q system (Millipore, St Quentin en Yvelines, France). 1-((ammoniooxy)methyl)-2-bromobenzene chloride (BBHA) was purchased from Interchim (Montluçon, France). Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) and trifluoroacetic acid (TFA) were purchased from Acros organics (Geel, Belgium).
[1,2-¹³C₂]-4-Hydroxy-2(E)-nonenal (¹³C-HNE). ¹³C-HNE was prepared according to our published method [18 (link)], with modifications. Ethyl [1,2-¹³C₂]-2-bromoacetate was converted to ethyl 4-hydroxy-2(E)-nonenoate by reacting consecutively with 4-chlorothiophenol, hydrogen peroxide, and n-heptanal as described [19 (link)]. The remaining steps were unchanged.
4-Hydroxy-2(E)-nonenal (HNE). HNE was prepared from ethyl 2-bromoacetate by the same method as for ¹³C-HNE.

All N-Fmoc- and orthogonally sidechain-protected amino acids, including Fmoc-S-acetamidomethyl-L-homocysteine, and peptide synthesis reagents were acquired from Chem-Impex Inc. (Wood Dale, IL, USA) and ChemPep. Fisher Scientific (Wellington, FL, USA) provided all of the solvents used, and Sigma-Aldrich (St. Louis, MO, USA) provided the riisopropylsilane (TIPS). Alfa Aesar (Ward Hill, MA, USA) provided the diisopropylethylamine (DIEA) along with the metal salts used in the experiment described. Trifluoroacetic acid (TFA) was purchased from Acros Organics (Fair Lawn, NJ, USA). The Rink Amide resin was bought from Gyros Protein Technologies (Uppsala, Sweden). Acridine orange nonyl bromide was purchased from Thermo Fisher (Eugene, OR, USA). Dialysis cassettes (2000 Da MW cutoff) were purchased from Thermo Fisher.

The synthetic peptides TAT (YGRKKRRQRRR), CTP (YGRRARRRRRR), R11 (RRRRRRRRRRR), TD (ACSSSPSKHCG), and their GABA‐conjugates were synthetized by SBS Genetech Co, Ltd. The peptide molecular weights were confirmed by mass spectrometry, 98% in purity. The lyophilized peptides were prepared and diluted in 0.9% NaCl before use.
γ‐aminobutyric acid (GABA) was provided by Beijing Gleckes Bio‐engineering Technology Co. Ltd. with a purity of 99%.
TAT‐choline acetyltransferase (TAT‐ChAT): The recombinant prokaryotic plasmid of pET15‐TAT‐ChAT was constructed to express the TAT‐ChAT fusion protein in Escherichia coli BL21 based on previous works (Fu et al. 2004, 2005). The purified TAT‐ChAT fusion protein (98% in purity) could effectively ameliorate the dementia symptom of the aged mice and the APP/PS1‐TgN transgenic mice in our other experiments. Lyophilized TAT‐ChAT was stored at −20°C, and dissolved in 0.9% saline solution before use.
Trifluoroacetic acid (TFA) was purchased from ACROS Organics (Belgian), extra pure with a purity of 99%. Formulated solutions (0.1%,0.5%,1%,2%,5%) were diluted with sterilized deionized water.

Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 132), and Serratia marcescens (ATCC 13880) were purchased from Carolina Biological Supply Company (Burlington, NC, USA) and stored as freezer stock cultures (nutrient broth:glycerol, 87.5∶12.5) at −80 °C.
Sinapinic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid (TFA) and acetonitrile were purchased from ACROS (Fair Lawn, NJ, USA). MALDI calibrants were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nutrient agar and nutrient broth were purchased from Carolina Biological Supply Company (Burlington, NC, USA).

Two batches of commercial freeze-dried Apis mellifera BV samples obtained by electrostimulation were used in this study, namely “BV-1” (Henan-Senyuan Biological Technology Co Ltd, China) and “BV-2” (Citeq biologics, Netherlands). Melittin ( $\ge 85$ % purity) was purchased from Sigma-Aldrich (UK). Nutrient agar (Oxoid Ltd., CM003), Nutrient broth (Oxoid Ltd., CM0001) and Phosphate-buffered saline (PBS) were supplied by Fisher Scientific (United Kingdom). Culture medium Luria–Bertani (LB) broth (Miller, L3152) and two stains, bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC₄(3)) and propidium iodide (PI), were purchased from Sigma-Aldrich (UK). HPLC grade water and acetonitrile (ACN) were from Chem-Lab (Belgium). Trifluoroacetic acid (TFA) was from Acros organics (Belgium). All other common reagents were of the appropriate purity from various suppliers.

Methanol (HPLC grade/Optima LC/MS grade) and acetonitrile (Optima LC/MS grade) were purchased from Fisher (Illkirch, France), while formic acid was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Ultrapure water was obtained using a Milli-Q system (Millipore, Saint-Quentin-en-Yvelines, France).
4-(2-((4-Bromophenethyl)dimethylammonio)ethoxy) benzenaminium dibromide (APEBA) was prepared and used as described by Eggink et al. [25 (link)]. 1-((Ammoniooxy)methyl)-2-bromobenzene chloride (BBHA) was purchased from Interchim (Montluçon, France). Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) and trifluoroacetic acid (TFA) were purchased from Acros Organics (Geel, Belgium).
Standard HHE, HNE, 4-hydroxynonanal (OHN), and ω-oxo-carboxylic acids (5-oxo-pentanoic acid (5-OPA), 6-oxo-hexanoic acid (6-OHA), 7-oxo-heptanoic acid (7-OHA), 8-oxo-octanoic acid (8-OOA), 9-oxo-nonanoic acid (9-ONA), 10-oxo-decanoic acid (10-ODA)) were synthesized in house according to previously published methods [32 (link),33 (link)]. All other standard aldehydes and other standard compounds were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France), except for 9-oxo-10,12-octadecadienoic acid (9-OxoODE), 9-Oxo-10,12,15-octadecatrienoic acid (9-OxoOTrE), and α-linolenic and eicosapentaenoic acid which were purchased from Bertin Bioreagent (Montigny-le-Bretonneux, France).