Fe3 nta agarose beads

Approximately 25 μg of peptides from HMEC stimulated by 10 ng/mL EGF for 10 min or without stimulation were spiked with 20 fmol/μL of heavy labeled peptides and then subjected to IMAC for phosphopeptide enrichment. The magnetic Fe³⁺-NTA agarose beads were prepared by replacing the Ni²⁺ ion on the magnetic Ni-NTA agarose beads (Qiagen, Valencia, CA) with Fe³⁺ through buffer exchange.^{31 (link)} Peptides were reconstituted in 50 μL IMAC binding/wash buffer containing 80% ACN and 0.1% TFA and incubated for 30 min with 20 μL of the 5% preconditioned bead suspension. After incubation, the beads were washed 4 times with 50 μL of wash buffer. Phosphorylated peptides were eluted from the beads with elution buffer containing 50% acetonitrile and 5% ammonia in 5 mM phosphate buffer (pH 8). The eluate was acidified with acetic acid to a pH of ~3.5 and concentrated to ~20 μL by vacuum centrifugation. Four μL of IMAC enriched samples was loaded onto the analytical column for LC-SRM analysis. The on-column peptide loading amounts were nearly equal to those in the later PRISM-SRM analysis to enable comparison of sensitivity and recovery.

The remaining 92% of the sample was further concatenated into 12 fractions before being subjected to phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC) as previously described (Mertins et al., 2018 (link)). Ni-NTA agarose beads (QIAGEN) were conditioned and incubated with a 10mM FeCl3 aqueous solution at RT for 1h to prepare Fe3+-NTA agarose beads. Peptides from each fraction were reconstituted in 80% ACN, 0.1% trifluoroacetic acid (TFA) and incubated with 10 μL of the Fe3+-IMAC beads at RT for 30 min. The bead solution was spun down at 1,000 g for 1 min. The supernatant containing unbound peptides was separated from beads and collected for subsequent glycopeptide enrichment. The beads were resuspended in 80% ACN (0.1% TFA) and loaded onto conditioned C18 stage tip. The tip was washed twice with 80% ACN (0.1% TFA) followed by 1% FA. The flowthroughs were collected and combined with the unbound peptides for subsequent glycopeptide enrichment. Peptides were eluted from beads to C18 stage tip with 70 μL of 500 mM dibasic potassium phosphate, pH 7.0 three times. The tip was then washed twice with 1% FA to remove salts. Peptides were eluted twice with 80% ACN (0.1% FA), dried down, and redissolved in 3% ACN, 0.1% FA prior to ESI-LC-MS/MS analysis.