Xbridge premier peptide beh c18

Final LC–MS/MS
analysis for method evaluation was performed using an Orbitrap Eclipse
mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to
an UltiMate 3000 LC system (Thermo Fisher Scientific, Germering, Germany).
For these short gradients (5, 10, and 15 min), a self-packed trap
column (XBridge Premier Peptide BEH C18, 2.5 μm, 150 μm
i.d., 3 cm length, 130 Å; Waters, Milford, MA, USA) and a self-packed
analytical column (XBridge Premier Peptide BEH C18, 2.5 μm,
75 μm i.d., 5 cm length, 130 Å; Waters, Milford, MA, USA)
were used. The mobile phases were as follows: for the (i) positive
ion mode, (A) 0.1% FA in H₂O and (B) 0.1% FA and 95% ACN
in water and for the (ii) negative ion mode, (A) 2.5 mM imidazole
and 3% IPA in water and (B) 2.5 mM imidazole, 3% IPA, and 95% ACN
in water. The loading solvent was in both cases mobile phase A. The
gradient was from 2 to 40% B in the corresponding minutes of the gradient
(5, 10, or 15 min) at a flow rate of 1.5 μL/min. Full MS scans
were acquired with 240,000 resolution. For the Thermo Scientific Pierce
HeLa protein digest standard, 500 ng of the sample was injected. In
the case of the digestion with the different enzymes, 1 μg of
HeLa digest was loaded on the column. For each condition (positive
and negative), each chromatogram time (5, 10, and 15 min), and each
enzyme (trypsin, GluC, LysC, and AspN), the sample was injected in
quadruplicate.

LC methods were optimized by using
both LTQ Orbitrap XL and Q Exactive HF mass spectrometers (Thermo
Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000
LC system (Thermo Fisher Scientific, Germering, Germany). A μ-Precolumn
C18 PepMap100 trap column (5 μm, 300 μm i.d., 5 mm length,
100 Å; Thermo Fisher Scientific, USA) and a self-packed analytical
column (XBridge Premier Peptide BEH C18, 2.5 μm, 75 μm
i.d., 18 cm length, 130 Å; Waters, Milford, MA, USA) were used
for the separation. In each injection, 1 μg of the E. coli digest and a mixture of iRT peptides (Biognosis
AG, Schlieren, Switzerland) at a 1× concentration were injected.
Mobile phase A consisted of the corresponding modifier (FA in the
positive mode and imidazole/piperidine in the negative mode) in Milli-Q
water, and mobile phase B consisted of 95% ACN (VWR Chemicals, Rosny-sous-Boix,
France) in water with the modifier at the same concentration as in
mobile phase A. For the sake of simplicity and readability, the concentration
of the modifier will be specified in each of the experiments. When
isopropanol was present in the mobile phase (1 to 5%), it was added
at the same concentration in both mobile phases. The loading solvent
was in all cases mobile phase A. The LC gradient was from 2 to 40%
B in 60 min at a 250 nL/min flow rate.