Microflex maldi tof

After global deprotection, supernatants collected in 15 mL Falcon tubes for each code were placed into a freezer for 1 hr; these tubes were then centrifuged (4000 g for 20 min at 4 °C) and decanted (repeated 3 times). Peptides were then dissolved with 60% ACN/H2O (20 μL, phosphopeptides were dissolved with 50% acetic acid) for MALDI analysis using THAP (250 mM in ACN) as the matrix. To prepare the MALDI plate (microScout Target MSP 96 target polished steel BC, part #8280800), 0.5 μL of sodium citrate (250 mM in H2O containing 0.1% TFA) was spotted on to the plate surface and allowed to dry. After drying, 1 μL of a 1:1 mixture of the peptide solution with a solution of sodium citrate:THAP (1:1) was spotted onto the plate and allowed to dry again before analysis. Data was obtained using a Bruker microflex MALDI-TOF (Billerica, MA, USA). The instrument was run on positive-ion reflector mode with a laser setting of 1,810 V and data averaged over 100 scans. Raw data was analyzed using FlexAnalysis and mMass (ver. 5.5, http://www.mmass.org/).

The protein samples were prepared on a ground steel MSP96 target (Bruker Daltonik, Germany) using HCCA (α-cyano-p-hydroxycinnamic acid Bruker Daltonik, Germany) as a matrix, according to the manufacturer’s recommendations. Protein Standard I (Bruker Daltonik GmbH, Germany; containing Insulin, Ubiquitin, Cytochrome C and Myoglobin) and Protein Standard II (Bruker Daltonik GmbH, Germany; containing Trypsinogen, Protein A and BSA) were used to calibrate the respective mass ranges. Mass spectrometry measurements were performed in a microflex MALDI-TOF (Bruker Daltonik GmbH, Germany). The spectra fell within the mass range 5–60 kDa.

In order to analyze the intact mass of the sfGFP or sfGFP-PA conjugate, the sample was desalted on a ZipTip C18 according to the manufacturer’s (Millipore Corporation) protocol. The first layer applied to a polished steel plate using sinapinic acid in absolute ethanol. The desalted sfGFP or sfGFP-PA conjugate was mixed with 1:1 of sinapinic acid in TA30 solution (0.1% Trifluoroacetic acid: acetonitrile = 7:3) and then applied to the first layer prior to mass analysis via Microflex MALDI-TOF (Bruker Daltonics; Bremen, Germany). The MALDI-TOF was calibrated using a Protein Standard II (20–90 kDa) before measurement according to the manufacturer’s instructions. The average masses of sfGFP, SP01, SP02, SP03, and SP04 were obtained by multiplying each area and its corresponding mass of all peaks and then dividing its average value by the average area of all peaks. The average numbers of conjugated PAs at SP01, SP02, SP03, and SP04 were obtained by using the molecular weight of the PA containing linker from the mass shift from sfGFP, sfGFP-azides, and sfGFP-PEG4-azides.

Peptides were purchased from JPT Peptide Technologies GmbH or synthesized in-house, as indicated in Table S2. Peptides were produced in-house by solid-phase synthesis with the 9-fluorenyl-methoxy carbonyl (Fmoc)-method (CEM-Liberty and Applied Biosystems) on PEG-PS preloaded resins (Merck, Darmstadt, Germany) as previously described (59 (link), 60 (link)) with the following alterations. After synthesis the peptides were washed with 50 ml dichloromethane (Roth), cleaved from the resins using 28.5 ml trifluoroacetic acid (Roth), 0.75 ml silane (Sigma-Aldrich, St. Louis, MO, USA) and 0.575 ml H₂O for 2.5 hours at room temperature (RT) and precipitated into pre-chilled tert-butylmethylether (Merck). The peptides were purified by reversed-phase high-performance liquid chromatography in a 10–70% acetonitrile gradient using a Jupiter 4 μm Proteo 90 Å LC column (Phenomenex) and an UltiMate 3000 Pump (Dionex) to a purity >90%. Their identities and molecular weights were verified by mass spectrometry (Microflex MALDI-TOF, Bruker).

MALDI TOF MS was performed essentially as reported previously [18 (link),19 (link)]. Bacterial samples were smeared very thinly on the MSP 96 target polished steel BC plate and MALDI matrix solution was added. A Microflex MALDI-TOF (Bruker Daltonik GmbH, Leipzig, Germany) bench-top mass spectrometer was used for identification of sample isolates [18 (link),19 (link),22 (link)] and for analysis of the MALDI-TOF mass spectra, we used the version 3.1 (Build 65) of the Bruker MALDI Biotyper Real Time Classification Classification software. To design the dendrogram for bacteria identification, we then used the MALDI Biotyper MSP creation method (Bruker Daltonics, Bremen, Germany) as reported previously [18 (link),19 (link),22 (link)].

To determine their monoisotopic masses, we desalted GLP1_16AzF, GLP1_19AzF, and GLP1_28AzF using a ZipTip C18 system according to the manufacturer’s protocol. The desalted peptides were mixed with a 1:1 (v:v) mixture of α-cyano-4-hydroxy cinnamic acid (HCCA) saturated TA30 (a solution comprising 30% acetonitrile and 0.1% trifluoroacetic acid), and applied to a polished steel plate, then subjected to mass characterization by microflex matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF; Bruker Daltonics, Bremen, Germany).
To determine their intact masses, we desalted GLP1_16HSA, GLP1_19HSA, and GLP1_28HSA on a ZipTip C18 system according to the manufacturer’s protocol. The desalted conjugates were mixed with a 1:1 (v:v) mixture of DHB matrix (20 mg/mL 2,5-dihydroxybenzoic acid in TA30), and applied to a polished steel plate, then subjected to mass characterization by microflex MALDI-TOF (Bruker Daltonics, Bremen, Germany). The mass spectrum of each sample was obtained using flexControl autoflex TOF/TOF software (Bruker Daltonics, Bremen Germany).