Nanolc ultra 1d

The processed samples were published by
Wang et al.^{12 (link)} and downloaded from the PRIDE
repository^{25 (link)} with the identifier PXD010154.
From this dataset, the chosen subset of samples consists of 106 MS
raw files of healthy tonsil tissues acquired from 3 different experiments
with identifiers P010747, P010694, and P013107. Briefly, the proteins
were digested with trypsin and analyzed by tandem MS coupled with
liquid chromatography (LC–MS/MS) using a Q Exactive Plus mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to
a nanoflow LC system (NanoLC-Ultra 1D+, Eksigent, USA) using a 110
min gradient, yielding 5,085,477 MS/MS spectra (Exp. P010747: 1,834,613
MS/MS spectra, Exp. P010694: 1,695,460 MS/MS spectra, Exp. P013107:
1,555,404 MS/MS spectra). MS1 scans were acquired at a resolution
of 70,000, and MS2 scans were acquired for up to 20 precursors after
HCD fragmentation. For more details on the data generation, please
refer to the original publication by Wang et al.^{12 (link)} A quality control of the raw data was performed using the
software tool viQC,^{26 (link)} and the results for
each used raw file can be found at the GitHub repositorya.

The proteins were determined through reverse‐phase high‐pressure mass spectrometry via NanoLC‐Ultra 1D (Eksigent) and TripleTOF 5600+ (AB Sciex) as previously reported.19 The raw EBI data were searched using the ProteinPilot software. To sort through the large number of regulated proteins, the Gene Ontology (GO) pathway database was accessed via the GO automatic annotation server (ANNEX).

Following digestion, Lys‐8/Arg‐10 labelled standard peptides for E‐cadherin, β‐actin and GAPDH were added at defined concentrations before C18 MicroColumn clean‐up. SRM analysis was performed on a TSQ Vantage (Thermo) coupled to a nanoLC Ultra1D+ (Eksigent). Peptides were separated on a column (0.075 × 100 mm, C18 ProntoSIL 200 3 μM, 200A) packed into a PicoTip Emitter (New Objective). Peptides were separated along a linear gradient of B (98% ACN, 0.1% FA) in A (2% ACN, 0.1% FA), running from 2 to 45% B in 40 min. The TSQ was run in scheduled SRM mode, detecting three peptides per protein with three transitions for the light and heavy forms. Target peptides were quantified by their L/H XICs in Skyline v.4.1. E‐cadherin was normalised to the average abundance of beta‐actin and GAPDH and exported to GraphPad Prism v7.03.