Flexanalysis 2

Mass analysis was conducted on a Bruker microflex™ bench-top MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) using dried-droplet method on a micro scout target (MSP96 target ground steel BC, Bruker Daltonics) with α-cyano-4-hydroxycinnamic acid (HCCA, Sigma-Aldrich, Handels GmbH, Vienna, Austria) as matrix. Flex Analysis 2.4 software (Bruker Daltonics, Bremen, Germany) was used for data processing.

Tear samples were collected from mice by application of sterile filter paper strips (n=5, Schirmer paper), and PACAP38 was measured using MALDI-TOF mass spectrometry. Aqueous solutions of the standard and tear samples were loaded onto the target plate (MTP 384 massive target T, Bruker Daltonics, Bremen, Germany) by mixing 1.0 μl of each solution with the same volume of a saturated matrix solution, prepared fresh every day by dissolving α-cyano-4-hydroxycinnamic acid in acetonitrile/0.1% trifluoroacetic acid (1/2, v/v). The mass spectrometer used in this work was an Autoflex II TOF/TOF (Bruker Daltonics) operated in the linear mode. Ions were accelerated under delayed extraction conditions (140 ns) in the positive ion mode with an acceleration voltage of 20.00 kV. The instrument uses a 337 nm pulsed nitrogen laser, model MNL-205MC (LTB Lasertechnik Berlin GmbH, Berlin, Germany). External calibration was performed in each case using the average masses of the Bruker Peptide Calibration Standard (#206195, Bruker Daltonics). Protein masses were acquired within a range of 1,000–8,000 m/z. Each spectrum was produced by accumulating data from 800 consecutive laser shots. Bruker FlexControl 2.4 software was used for control of the instrument and Bruker FlexAnalysis 2.4 software for spectrum evaluation.

Mass spectra were recorded using the autoflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Germany) equipped with a pulsed N₂ laser (337 nm) in a positive reflectron mode. Ions formed by a laser beam were accelerated to 20 keV kinetic energy. The final spectra were obtained accumulating 1500 single laser shot spectrum.
The solution (50 mg/mL) of 2,5-dihydroxybenzoic acid (DHB) in aqueous acetonitrile was used as a matrix. A sample solution in 0.1% TFA in water was mixed with the same volume of matrix solution. About 1 μL of the resulting solution was deposited on the 384 ground steel target plate and allowed to dry before being introduced into the mass spectrometer. External calibration in positive mode was done using Peptide Calibration Standard II (Part No. 222570, Bruker Daltonics, Germany). Mass accuracy of about 10 ppm was usually achieved. Mass spectra were processed using FlexAnalysis 2.4 software (Bruker Daltonik GmbH, Germany). Rapid identification of proteins based on a peptide mass fingerprint search through the Swiss-Prot database was done using Mascot Server (Matrix Science Inc., USA).

MALDI-TOF mass spectrometry analyses were performed using Ultraflex TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Mass spectra were recorded in linear mode of positively charged ions in the m/z range of 5–20 kDa using 2,5-dihydroxybenzoic acid (20 mg/ml, acetonitrile/0.1% TFA 1:1, v/v) as a matrix. The mass spectrometry data were processed using Bruker Daltonics Flex Analysis 2.4 software.

100 µL of a 50 µg/mL solution of ^natLu-PSMA I&T in a 1 mL Eppendorf tube was subjected to external irradiation using a ¹³⁷Cs IBL 437L irradiator (CIS US Inc., NY, USA) at a dose rate of about 5 Gy/min for 100 min reaching a dose of 500 Gy. The sample was purified by HPLC (injection volume 50 µL) using HPLC method A and the peaks of the impurity and the main peak due to ^natLu-PSMA I&T were collected and analysed by MALDI-TOF MS.
Mass analysis was conducted on a Bruker microflex® bench-top MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) using dried-droplet method on a micro scout target (MSP96 target ground steel BC, Bruker Daltonics) with α-cyano-4-hydroxycinnamic acid (HCCA, Sigma-Aldrich, Handels GmbH, Vienna, Austria) as matrix. Flex Analysis 2.4 software (Bruker Daltonics, Bremen, Germany) was used for data processing.

Protein identities were analyzed using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS), as described by Lu et al.^{51 (link)}. Mass spectra were processed using FlexAnalysis 2.4 software (Bruker Daltonics, Bremen, Germany). Peptide masses were searched against a comprehensive nonredundant protein sequence database (NCBInr 20130524 version with 25805290 sequences and 8915431356 residues; SwissProt 2013_05 version with 540052 and 191770152 residues) employing BioTools 3.0 software (Bruker Daltonics) in combination with the MASCOT program^{22 (link)}. Protein identities were further confirmed through MALDI TOF/TOF MS/MS analysis by using a Bruker UltraFlex III MALDI-TOF/TOF MS (Bruker Daltonics) equipped with a delayed extraction ion source. The metastable ions generated by laser-induced decomposition (LID) in the laser-induced forward transfer (LIFT) mode (Bruker Daltonics) was analyzed. The MS/MS spectra were searched against the NCBInr database (NCBInr 20130524 version with 25805290 sequences and 8915431356 residues; SwissProt 2013_05 version with 540052 and 191770152 residues) using Bio-Tools 3.0 software (Bruker Daltonics) in combination with the MASCOT program.