Nanoelectrospray emitter tip of 10 m

The peptides were initially enriched on a nano-trap column (C18, 2 cm, 5 µ, 100 Å, Agilent), followed by elution on to analytical column (ZORBAX 300SB-C18, 0.1 × 150 mm, 3.5 µ, Agilent). The peptides were sprayed using a nanoelectrospray emitter tip of 10 µm (Bruker, Germany), using 0.1% formic acid (FA) in water as solvent A and 0.1% FA in ACN as solvent B. The peptides were loaded onto the trap column using 97% solvent A, followed by resolution on the analytical column using a linear gradient of 2–45% solvent B for 55 min at a constant flow rate of 300 nL/min. The data were acquired in data-dependent acquisition mode, subjecting the six most intense ions in each survey scan to MS/MS analysis within an m/z range of 400–2200. The collision-induced dissociation (CID) method was used for precursor fragmentation, and the precursor ions selected for MS/MS fragmentation were excluded after every three spectra. The absolute threshold for precursor ions per 1000 summations was 1200 counts.

The fractionated peptides were reconstituted in 0.1% FA, followed which the proteins were identified using captive spray-Maxis-HD qTOF (Bruker Daltonics, Germany) MS with high mass accuracy and sensitivity. The peptides were initially enriched on a nano-trap column (C18, 2 cm, 5 µ, 100 Å, Agilent), followed by elution on to analytical column (15 cm, 3 µ, 100 Å, Agilent). The peptides were sprayed using Nano electrospray emitter tip of 10 µm (Bruker, Germany) using 0.1% FA in water as solvent A, and 0.1% FA in ACN as solvent B. The peptides were loaded onto the trap column using 97% solvent A, followed by resolution on the analytical column using a linear gradient of 5–30% solvent B for 70 min at a constant flow rate of 400 nl/min. The data were acquired in data-dependent acquisition mode subjecting the six most intense ions in each survey scan to MS/MS analysis within a m/z range of 400–2200. The collision-induced dissociation (CID) method was used for precursor fragmentation, and the precursor ions selected for MS/MS fragmentation were excluded after every three spectra. The absolute threshold for precursor ions per 1000 summations was 1200 counts^{88 (link),90 (link)}.

The fractions were analyzed on maxis-HD (Bruker, Bremen, Germany) interfaced with Nano-LC (Bruker, Bremen, Germany). Peptides were initially enriched on a reversed phase liquid chromatography (RPLC) pre-column (2 cm, 5 µ – 100 Ǻ), followed by separation on an analytical column (15 cm, 3 µ – 100 Ǻ) (Agilent). The peptides were sprayed using nano electro spray emitter tip of 10 µm (Bruker, Bremen, Germany). The solvent system used includes 0.1% aqueous formic acid as solvent A and 100% acetonitrile, 0.1% formic acid as solvent B. The peptides were loaded on the trap column using 97% solvent A, followed by separation on the analytical column using a linear gradient of 5–30% solvent B for 70 min at a constant flow rate of 0.400 µL/min. The spray voltage and heated capillary temperature were set to 2.0 kV and 220 °C, respectively. The data was acquired in data-dependent acquisition mode subjecting the six most intense ions in each survey scan to MS/MS analysis within the m/z range of 400–2200. The precursor fragmentation was carried out using collision-induced dissociation (CID) as the activation method. The precursor ions selected for MS/MS fragmentation were excluded after every three spectra. The absolute threshold for precursor ions per 1000 summations was 1200 counts^{32 (link)}.