Fe nta phosphopeptide enrichment kit

Phosphopeptide enrichment was performed on the TMT-labelled peptide mixture using the Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, MA, USA) based on the manufacturer’s instructions. Briefly, the lyophilised TMT-labelled peptides were resuspended with binding buffer before loading onto a spin column containing pre-equilibrated Fe-NTA. Enrichment was performed at RT for 30 min before washing three times with the washing buffer. Phosphopeptides were subsequently eluted from the beads and dried using a vacuum concentrator before resuspension with 0.1% formic acid. The peptide concentration was determined using the NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

For digestion, 500 µg of proteins were trypsin digested as described in Luu et al. (2017) (link) and cleaned up with an Oasis HLB 1cc vac cartridge (Waters). After cleanup, phosphopeptides were sequentially enriched for monophosphorylated peptides followed by multi-phosphorylated peptides using TiO₂ and IMAC, respectively (Figure 1) (Thingholm et al., 2008 (link)). First, TiO₂ enrichment was performed using Titansphere Phos-TiO Spin Tip kit (3 mg/200 µl) (GL Sciences) according to manufacturer’s instructions. Any uncaptured phosphopeptides in the flow through were cleaned up with an Oasis HLB 1cc vac cartridge and further enriched in a second IMAC fraction using the high-select Fe-NTA Phosphopeptide enrichment kit (ThermoFisher) according to manufacturer’s instructions.

A 2D affinity chromatography was conducted for phosphopeptide enrichment for each of the 9 fractions. In the first step peptides were subjected to an immobilized metal affinity chromatography using a Fe-NTA phosphopeptide enrichment kit according to the manufacturer’s protocol (#88300, Thermo Fisher Scientific). Eluted phosphopeptides were acidified by TFA to final concentration of 2.5% and dried by vacuum centrifugation. All flow-troughs after sample loading were pooled, dried by vacuum centrifugation and stored for subsequent enrichment step by metal oxide affinity chromatography (MOAC) using the TiO₂ phosphopeptide enrichment spin tips (#88303, Thermo Fisher Scientific). Spin tips were equilibrated by washing with Buffer A (80% ACN/0.4% TFA) first and then with Buffer B (57% ACN/0.3% TFA/25% lactic acid). Peptides were suspended in Buffer B and applied to the spin tips. After reapplying samples, spin tips were again washed with Buffer B and three times with Buffer A before peptides were eluted with 1.5% NH₄OH first and then with 5% pyrrolidine. Eluted samples were acidified with TFA to a final concentration of 1.25% and dried by vacuum centrifugation. All IMAC and TiO₂ elution fractions were cleaned up prior to MS analysis using graphite spin columns (#88302, Thermo Fisher Scientific) according to the manufacturer’s protocol.

All qualified profiling data were processed at firmiana platform against the human RefSeq protein database (updated on July 4, 2013) in the National Center for Biotechnology Information (NCBI). Owing to the definite volume of the samples of the early ESCC cohort, only 145 samples (from 41 ESCC patients) were found to be adequate.
The phosphoproteome samples were prepared by Fe-NTA Phosphopeptide Enrichment Kit (Thermo, Catalog: A32992) according to the manufacturer’s instructions. Briefly, 2 mg peptides were resuspended in 200 μL binding/wash buffer and loaded to the equilibrated spin column. The resin was mixed with the sample by gently tapping. The mixture was incubated for 30 min and centrifuged at 1000 × g for 30 s to discard the flowthrough. The column was then washed with 200 μL of binding/wash buffer and centrifuged at 1000 × g for 30 s three times and washed with 200 μL of LC-MS grade water one more time. The phosphopeptide was eluted with 100 μL of elution buffer and centrifuged at 1000 × g for 30 s two times. Phosphopeptides were dried down for LC-MS/MS analysis.

The label-free quantitative proteomics method was used for MS by Shanghai Bioprofile Technology Co., Ltd. Cell pellets were harvested, and total protein was extracted using SDT cell lysis reagent. Digested peptides were desalted using peptide desalting spin columns and lyophilized under vacuum. Peptide concentrations were measured using a Nanodrop. When performing the MS for phosphorylated proteins, the peptide solution was lyophilized under vacuum, and phosphorylated peptides were enriched with an Fe-NTA Phosphopeptide Enrichment Kit (Thermo, A32992); enriched phosphorylated peptides were collected according to the kit procedure for mass spectrometry analysis. An appropriate amount of enriched peptides for each sample was separated using a nanoliter flow rate Easy nLC 1200 chromatographic system (Thermo Scientific). MSFragger 3.4 software was used to retrieve data from UniProt Protein Data Bank (UniProt Homo sapiens (Human) [9606]-203800-202201.fasta). After comparison, a log2 (fold change) ≥ 1.5 (total proteins) or 2 (phosphorylated proteins) and P ≤ 0.05 were considered to indicate significantly different expression of modifier sites.

All qualified profiling data were processed at firmiana platform against the human RefSeq protein database (updated on 04-07-2013) in the National Center for Biotechnology Information (NCBI). Owing to the definite volume of the samples, only 111 samples (from 49 EDC patients) were found to be adequate.
The phosphoproteome samples were prepared by Fe-NTA Phosphopeptide Enrichment Kit (Thermo, Catalog: A32992) according to the manufacturer’s instruction. Briefly, 2 mg peptides were resuspended in 200 μL binding/wash buffer and loaded to the equilibrated spin column. The resin was mixed with the sample by gently tapping. The mixture was incubated for 30 min and centrifuged at 1000g for 30 s to discard the flowthrough. The column was then washed by 200 μL of binding/wash buffer and centrifuged at 1000g for 30 s for 3 times and washed by 200 μL of LC–MS grade water for one time. The phosphopeptide was eluted by adding 100 μL of elution buffer and centrifuged at 1000g for 30 s 2 times. Phosphopeptides were dried down for LC–MS/MS analysis.