b'High-Select High-SelectHSA/Immunoglobulin Top 14M Serum FT E Serum FT E 15,000 Phosphopeptides Phosphospecicity 100Phosphopeptide identications 12,000 80 Phosphospecicity (%) 9,000 606,000 403,000 200 0High-Select Fe-NTA kit Supplier AFigure 6. High-Select protein depletion spin columns removeFigure 8. Phosphopeptide enrichment from nocodazole-treated proteins to enable visualization of low-abundance proteins. HumanHeLa cell digests. 1 mg of digest was enriched using 25 L of Fe-NTA serum (1020 L) was loaded onto each resin and processed accordingmagnetic beads before LC-MS analysis (n = 3). Enrichment using to protocols. Total protein in the depleted fractions was estimatedThermo Scientific High-Select Fe-NTA Magnetic Agarose beads had using the Thermo Scientific Pierce BCA Protein Assay Kit. The totalhigher phosphopeptide identifications and equivalent phosphospecificity amount of albumin in the depleted fractions was determined using the(97%) compared to another suppliers resin. AssayMax Human Albumin ELISA Kit. FT = flowthrough (i.e., depleted sample); E = eluate (i.e., proteins that were bound by the resin, plus stripped affinity antibodies of the column). With the top 14 proteins removed, low-abundance proteins are now visible in each depleted sample lane (FT).9,000 Total unique peptides 99 100PhosphopeptidesSelectivity (%)806,0456,000 70No. of peptides60 Selectivity (%)3,932403,000200 088300 (8 g) A32992 (33 g)Figure 7. The High-Select Phosphopeptide Enrichment Kit (Cat. No. A32992) improves selectivity and yield over the Pierce Fe-NTA Phosphopeptide Enrichment Kit (Cat. No. 88300). The average selectivity for phosphopeptides is 99% with a 3- to 4-fold improvement in peptide yield. Protein digests (1 mg) from nocadazole-arrested HeLa cells were used for the enrichment according to instructions. Eluted peptides were analyzed using a Thermo Scientific EASY-Spray C18 LC Column (3 m, 150 mm) on a Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer.Mass spectrometry thermofisher.com/proteinbiology 95'