Anchorchip target plate

Although all centers shared very similar instrumentation (Table 1), each MSI experiment was preceded by the measurement of a centrally distributed dilution series of a bovine serum albumin digest (Pierce™ BSA Protein Digest, # 88341, Thermo Fisher) in order to monitor potential intra- and intercenter differences in instrument performance. This concentration series was prepared centrally (ESM, Protocol S1) and shipped to all remaining partners on dry ice. Finally, each local laboratory mixed each dilution again 1:1 with their locally prepared matrix (7 mg/mL alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.2% trifluoroacetic acid); 2 μL of each dilution was then pipetted onto an AnchorChip target plate (Bruker Daltonik) leading to absolute amounts in the spotted volume in the pico- to femtomole range.
For each droplet, 2500 spectra were acquired in random walk mode (50 spectra per step) over an area with a 500-μm diameter with the same settings as for the tryptic peptide MSI experiments (see below).

Urine samples were centrifuged at 2,100 g for 30 minutes at 4°C to remove cellular debris. The supernatant was recovered, adjusted to neutral pH with 1 M NH₄HCO₃, aliquoted, and immediately frozen at −80°C until processing.
Samples were thawed and pre-fractionated using Dynabeads RPC18 (Invitrogen, Breda, The Netherlands). Samples were processed in duplicate following the manufacturer’s protocol but modified for optimization purposes, as described previously [22] (link). Fifteen microliters of peptide eluate were obtained from each sample, diluted 1∶5 with LC-grade water (Lab-Scan, Gliwice, Poland), and mixed 1∶2 with matrix solution (1.84 mg/ml 2,6-dihidroxyacetophenone, 20% acetonitrile, 40 mmol/l ammonium citrate dibasic). Of the resulting mixture, 1µl was spotted in duplicate onto the sample anchor spots of an AnchorChip 600/384 target plate (Bruker Daltonics, Bremen, Germany) and allowed to air-dry at room temperature to let the matrix crystallize. Four spots of each sample were analyzed by MALDI-TOF MS. ClinProt Peptide Calibration Standard I (Bruker Daltonics), a commercially available mixture of protein/peptide calibrators, was mixed 1∶1 with matrix solution and 0.4 µl were deposited onto calibrant anchor spots of the AnchorChip target plate for instrument calibration.

MALDI-TOF was used to confirm the molecular mass of the designed collagen (Bruker Daltonics UltrafleXtreme, USA). 1 μL of 1 mg mL⁻¹ sample was dropped in a 600 μm AnchorChip target plate (Bruker) and allowed to dry, using 5 mg mL⁻¹ 2,5-dihydroxyacetophenone, 25% (v/v) ethanol, 1.5 mg mL⁻¹ diammonium hydrogen citrate as the matrix in linear mode, BSA was used for external calibration. Or using 10 mg mL⁻¹ sinapinic acid in 50% acetonitrile, 0.1% TFA as the matrix. 1 μL of the sample was applied onto a plate with apo-myoglobin as the internal standard. Data was acquired with AB Sciex 4800 at positive linear mid-mass mode.

MALDI-TOF-MS in reflection positive-ion mode was used for the serum N-glycans profiling (17 (link)). 1 μL of glycan samples was mixed with 1 μL of matrix containing 5 mg/mL super-DHB with 1 mM NaOH in 50% ACN on the AnchorChip target plate (Bruker Daltonics, Bremen, Germany) and the mixture was dried by air for 2 h. The N-glycomes were detected using a rapifleXtreme MALDI-TOF mass spectrometer with a Smartbeam-3D laser, controlled by flexControl 4.0 (Bruker Daltonics). A peptide calibration standard (Bruker Daltonics) was used for the calibration of the instrument. The mass range was set from m/z 1000 to m/z 5000. Laser shots were accumulated 5000 times for each spectrum. We chose an automatic acquisition mode and random walk pattern at a laser frequency of 5000 Hz for the acquisition of sample spectra (17 (link)).

Firstly, 1 μL of saturated α-cyano-4-hydroxycinnamic acid (2 mg/mL) in 0.1% TFA with ACN (2:1) was coated on each spot of the anchor-chip target plate (Bruker, Germany). The dried peptides were resuspended with 2 μL of 0.1% TFA with ACN (2:1) and then 1 μL was added onto the dried anchor spots. After the spot dried, the anchor spot was briefly washed with 0.1% TFA and subsequently recrystallized with 1 μL of recrystallization solution (ethanol: acetone: 0.1% TFA = 6:3:1). The mass spectra, ranging from 700 to 3000 Da, were determined using the reflector mode of Bruker Autoflex III Series High-Performance MALDI-TOF & TOF-TOF systems after calibration with an external peptide calibration standard (Bruker, Germany) was conducted. Spectra from 500 shots at different positions on the target plates were combined to generate a peptide-mass fingerprint. The obtained peptide masses were searched against the National Center for Biotechnology Information (NCBI) protein database of medaka (NCBI:txid8090) by using the MASCOT search engine [51 (link), 52 (link)]. The selected database consisted of 38,099 sequences and 22,982,484 residues on 12 Oct 2015.

Mass spectrometry grade water, trifluoroacetic acid (TFA), sodium hydroxide, dithiothreitol (DTT) and ammonium bicarbonate were purchased from Fisher Scientific (Sunnyvale, CA, USA). Mass spectrometry-grade acetonitrile (ACN) and formic acid (FA) were purchased from Fluka Analytical, by Merck (Kenilworth, NJ, USA). Hydrogen chloride was purchased from Univar (Downers Grove, IL, USA). Equipment used in MALDI-TOF mass spectrometry including Anchorchip target plate, a-cyano-4-hydroxy-cinnamic acid matrix and peptide calibrant standard II were all purchased from Bruker (Bremen, Germany).