Ziptip pipette tip

Protein concentrations were measured using the BCA Protein Assay (Thermo
Fisher Scientific). Reduction, alkylation, and trypsin digestion were performed
as previously described (32 (link)). For
phosphotyrosine peptide enrichment, peptide immunoprecipitation was performed
using p-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as
previously described (32 (link)). Prior to
immunoprecipitation, a 5 pmol fraction of synthetic phosphopeptide LIEDAEpYTAK
was added to each individual replicate and time point sample as a normalization
standard to correct for any variation in mass spectrometer sensitivity between
individual replicates. Samples were then desalted using ZipTip pipette tips (EMD
Millipore, Billerica, MA) according to the manufacturer’s
instructions.

Each of the eight synthetic peptides (2 pmol/μL) was incubated with WT ACE, NKO ACE, or without ACE (only buffer was added) in 100 μL of HEPES buffer (50 mM HEPES, 300 mM NaCl, and 400 mM Na₂SO₄; pH 8.15) for 30 min at 37 °C on an Eppendorf Thermomixer R at 300 rpm. The enzymatic reaction was stopped by addition of trifluoroacetic acid (TFA) for a final pH of <4.0. The samples were desalted and concentrated on ZipTip Pipette Tips (C18, 10 μL loading capacity, 0.6 μL bed format, EMD Millipore Corporation), eluted with 1.5 μL of 0.1% TFA/70% ACN, and spotted directly onto a MALDI-TOF-MS target plate. Matrix solution, α-cyano-4-hydroxycinnamic acid (CHCA, 0.5 μL, ProteoChem), prepared by dissolving 10 mg of the pure chemical in 1 mL of 0.1% TFA/70% ACN mixture, was added immediately to each sample spot. The samples were analyzed on a Simultof 200 Combo MALDI-TOF mass spectrometer (Virgin Instruments) operated in reflectron mode, and spectra of positively charged ions were acquired at 500 laser shots per spectrum. A stock solution of peptide standards was used for instrument mass calibration. It contained bradykinin fragment 2–9, substance P, neurotensin, antioxidant peptide (PFTRNYYVRAVLHL), amyloid B protein fragments 1–28 and 12–28, and ACTH fragments 1–24 and 18–39, at a concentration of 1 pmol/μL for each peptide in 0.1% TFA.