Chip cube

Each gel plug was digested with 10 μL of 5 mM ammonium bicarbonate with 10% ACN containing 100 ng trypsin (Promega) for 16 hours at 37°C. Peptides were extracted sequentially with 1% formic acid (FA), 50% ACN/0.1% FA, and ACN, and the combined extracts were concentrated by centrifugal evaporation and diluted in 6 μL 3% ACN/0.1% FA. Vacuum concentrated samples were resuspended in 0.1% FA in 2% ACN to a total volume of ~8 μL. LC-ESI-IT MS/MS was performed using an online 1100 series HPLC system (Agilent Technologies) and HCT Ultra 3D-Ion Trap mass spectrometer (Bruker Daltonics). The LC system was interfaced to the MS using an Agilent Technologies Chip Cube operating with a ProtID-Chip-150 (II), which integrates the enrichment column (Zorbax 300SB-C18, 4 mm, 40 nL), analytical column (Zorbax 300 SB-C18, 150 mm × 75 μm), and nanospray emitter. 5 μL samples were loaded onto the enrichment column set at a flow rate of 4 μL/min in Mobile Phase A (0.1% FA in 2% v/v ACN) and resolved with 1–30% gradient of Mobile Phase B (0.1% FA in 98% w/v ACN) over 32 minutes at 300 nL/min. Ionizable species (300 < m/z < 3,000) were trapped and the two most intense ions eluting at the time were fragmented by collision-induced dissociation. Active exclusion was used to exclude a precursor ion for 30 seconds following the acquisition of two spectra.

Peptides mixtures were analyzed by LC-MSMS on a 6520 Accurate-Mass Q-Tof LC/MS System (Agilent Technologies, Palo Alto, CA, USA) equipped with a 1200 HPLC System and a chip cube (Agilent Technologies). Details are reported in Supplemental Methods.

Samples were treated in heterogeneous phase with different pre-treatments (either incubation with 6M urea, or extraction with TFA 0.1%, acetonitrile 10%, or extraction with RIPA buffer, or extraction with CH₃CL₃/CH₃OH [6: 3; v/v]). This phase was followed by enzymatic digestion with trypsin at 37°C for 16 hours, purification using a reverse-phase C18 Zip Tip pipette tip (Millipore), and nano LC-MS/MS analysis on a CHIP MS 6520 QTOF equipped with a capillary 1200 HPLC system and a chip cube (Agilent Technologies, Palo Alto, CA) [72 ]. Raw data were used for protein identification with a licensed version of MASCOT software (www.matrixscience.com) version 2.4. with 10 ppm MS tolerance and 0.6 Da MS/MS tolerance; peptide charge from +2 to +3. No fixed chemical modification was inserted, but possible oxidation of methionines, deamidation at asparagines and glutamines, and the addition of hydroxylation on prolines and lysines were considered as variable modifications to query the SwissProt database, with Homo sapiens as a taxonomy restriction.

2‐AA‐labeled N‐glycans were analyzed with an Agilent 6300 MS LC‐MS system consisting of a 1200 LC system connected to an Agilent chip cube with integrated ion source serving as the interface to a an Agilent 6300 ion trap. Briefly, samples (6 µL per injection, corresponding to 0.8 µL CSF starting material) were separated on a 150 mm Zorbax‐300SB‐C18 chip column with integrated trap column (Agilent, G 4240‐62010) with increasing concentration of acetonitrile (0.2% formic acid in H₂O in pump system A and 0.2% formic acid in acetonitrile in pump system B). A flow rate of 3 µL·min⁻¹ was used, and the gradient was performed in a stepwise manner: 0–1 min, 1% buffer B; 1–2 min, 1–4.5% buffer B; 2–34 min, 4.5–8.5% buffer B; 35–38 min, 95% buffer B; 40–50 min, 1% buffer B.

A 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA) equipped with a 1200 HPLC system and a chip cube (Agilent Technologies, Santa Clara, CA, USA) was used to analyse the peptide mixtures. 1 μL of the peptide mixture was injected by an autosampler and desalted at a flow rate of 4 μL/min in a 40 nL enrichment column with 0.1 % HCOOH as eluent. A C18 reverse-phase capillary column (75 mm × 43 mm, 100A) included into an Agilent Technologies chip (Santa Clara, CA, USA) was used to fractionate the sample at a flow rate of 400 nL/min, with a linear gradient of eluent B (0.1 % HCOOH in 95 % ACN) in A (0.1 % HCOOH in 2 % ACN) from 5 to 80 % in 50 min.
Data-dependent acquisition of one MS scan in the mass range m/z 300–2400 was carried out, followed by an MS/MS scan of the five most abundant ions in each MS scan. MS/MS spectra were measured automatically when the MS signal was greater than the threshold of 5000 counts. Charge ions preferably isolated were double, triple, and quadruple, and they were fragmented over singly charged ions.

Protein band corresponding to ~62kDa were excised from the gel, destained and subjected to trypsin or chymotrypsin digestion and resulting peptide extracts were reduced by vacuum centrifugation. LC-eSI-IT MS/MS was performed using an online 1100 series HPLC system (Agilent Technologies) and HCT Ultra 3D-Ion-Trap mass spectrometer (Bruker Daltonics). The LC system was interfaced to the MS using an Agilent Technologies Chip Cube operating with a ProtID-Chip-150 (II), which integrates the enrichment column (Zorbax 300SB-C18, 4 mm, 40 nL), analytical column (Zorbax 300 SB-C18, 150 mm x 75 μm), and nanospray emitter. Fragmented ARX peptides were separated by reverse phase HPLC using C-18 column and 0–30% acetonitrile gradient. MS and MS/MS spectra were subjected to peak detection and de-convolution using DataAnalysis (Version 3.4, Bruker Daltronics). Compound lists were exported into BioTools (Version 3.1, Bruker Daltonics) and submitted to Mascot (Version 2.2) LC-ESI-IT-MS/MS analysis was performed as a fee for service by Adelaide Proteomics Centre, University of Adelaide, Australia.

Mass spectrometric analysis by infusion was performed as described previously (Takeuchi et al, 2012). Briefly, dried samples were resuspended in 0.1% formic acid in water, spin‐filtered, and injected into an Agilent 6340 ion‐trap mass spectrometer with a nano‐HPLC CHIP–Cube interface at a rate of 18 μl/h. The MS peaks for MS/MS were chosen manually, and the data were analyzed using Agilent ChemStation data analysis software. The masses of EGF repeats with different charge states were deconvoluted and shown on the top of peaks in HPLC profiles.