1260 infinity nhplc stack

Peptide digests (8 μL each) were injected onto a 1260 Infinity nHPLC stack (Agilent Technologies), and separated using a 75 μm inside diameter x 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system runs in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a Nanospray Flex^TM ion source (Thermo Fisher Scientific); all data were collected in collision-induced dissociation mode. The nano-High Performance Liquid Chromatography is configured with binary mobile phases that includes solvent A (0.1%FA in ddH₂O), and solvent B (0.1%FA in 15% ddH₂O / 85% ACN), programmed as follows; 10min @ 5%B (2μL/ min, load), 90min @ 5%-40%B (linear: 0.5nL/ min, analyze), 5min @ 70%B (2μL/ min, wash), 10min @ 0%B (2μL/ min, equilibrate). Following each parent ion scan (300-1200m/z @ 60k resolution), fragmentation data (MS2) were collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.

Peptide digests (8 μL each) were injected onto a 1260 Infinity nHPLC stack (Agilent) and separated using a 75 micron I.D. × 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system ran in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nanoelectrospray source (Thermo Fisher Scientific), and all data were collected in CID mode. The nHPLC was configured with binary mobile phases that included solvent A (0.1% formic acid in ddH₂O) and solvent B (0.1% formic acid in 15% ddH₂O/85% ACN) programmed as follows: 10 min @ 0%fet alB (2 μL/min, load), 90 min @ 0%−40%B (0.5 nL/min, analyze), 15 min @ 0%B (2 μL/min, equilibrate). Following each parent ion scan (350–1200 m/z @ 60k resolution), fragmentation data (MS2) was collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.

Dried peptides were reconstituted in 16 µL of 0.1% formic acid (FA). 8 µL of each sample was injected onto a 1260 Infinity nHPLC stack (Agilent Technologies, Santa Clara, CA, USA), and separated using a 75 micron I.D. × 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system runs in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nano-electrospray source (Thermo Fisher Scientific, Waltham, MA, USA), and all data were collected in CID mode. The nHPLC was configured with binary mobile phases that included solvent A (0.1%FA in ddH2O), and solvent B (0.1% FA in 15% ddH₂O / 85% ACN), programmed as follows; 10 min at 2% solvent B; 90 min at 5-40% solvent B; 5 min at 70% solvent B; 10 min at 0% solvent B. Following each parent ion scan (300-1200m/z @ 60k resolution), fragmentation data (MS2) was collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.