Liquid chromatography separation of a 2 μg sample was achieved by reversed-phase chromatography using a NanoLC Ultra 2D HPLC (Eksigent) equipped with a nanoflex cHiPLC set to 37°C. A 90-minute gradient was used for peptide separation, as described (7 (link)), followed by elution directly into a 5500 QTrap (AbSciex). Peptide precursors were selected in quadrupole 1 (Q1), fragmented in q2, and the top 3–4 transitions were selected for monitoring in Q3. All Q1 and Q3 masses were measured at unit resolution. A 7-minute scheduling window was applied with a 1.5-second cycle time. Method development and peak analysis were done using Skyline software (18 (link)).
Nanolc ultra 2d hplc
Manufactured by AB Sciex
The NanoLC Ultra 2D HPLC is a high-performance liquid chromatography system designed for the separation and analysis of complex samples. The system features a dual-pump configuration that enables two-dimensional separation, providing enhanced resolution and separation capabilities. The NanoLC Ultra 2D HPLC is a versatile instrument suitable for a wide range of applications requiring high-sensitivity and high-resolution chromatographic separations.
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4 protocols using nanolc ultra 2d hplc
1
Targeted Peptide Quantification by LC-MS/MS
2
Quantitative Serum Protein Analysis
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Serum samples from patients were prepared for quantitative analysis as previously described (7 (link)). Briefly, whole blood serum (40 μL) was filtered using a 0.22-μm filter and then depleted of the top seven most abundant serum proteins using a 4.6 × 100-mm human multiple affinity removal system column (MARS; Agilent Technologies). A fixed amount of the proteotypic reference standard was spiked into each protein sample, before trypsin digestion. A 90-min liquid chromatography gradient then resolved 2 μg of purified tryptic peptides on a NanoLC Ultra 2D HPLC (Eksigent) column, equipped with a nano-flex cHiPLC set at 37 °C. Finally, the ratio of test to reference peptides was analyzed after resolution on a QTrap 5500 model triple quadrupole mass spectrometer (Sciex) with Q1 as a precursor ion mass filter, q2 to fragment, and Q3 to select the top three or four fragment ions for quantitation.
3
Nano-LC-MS/MS Proteomics Protocol
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One microliter of sample was injected
onto a NanoLC-Ultra 2D HPLC (Eksigent, Dublin, CA) system equipped
with a 5 μL injection loop. Separation was performed with a
capillary column (75 μm ID, 10 cm length, 15 μm orifice)
created by hand packing a commercially available fused-silica emitter
(New Objective, Woburn MA) with 5 μm Luna C18 bonded separation
media (Phenomenex, Torrance, CA). The flow rate was 1000 nL/min for
5.5 min, then decreased to 300 nL/min with a 40 min linear gradient
of 2 to 30% CH3CN in 5 mM NH4OAc aqueous buffer
(pH 6.8), followed by a 5:95 buffer/CH3CN hold for 10 min
and a 5 min re-equilibration at 1000 nL/min 98:2 buffer/CH3CN. The injection valve was switched at 6 min to remove the sample
loop from the flow path during the gradient. Samples were analyzed
by nanoelectrospray using an LTQ Orbitrap Velos instrument (Thermo
Scientific, Waltham, MA). The nanoelectrospray source voltage was
set at 1.6 kV, and the capillary temperature was 350 °C. The
ion focusing and transfer elements of the instrument were adjusted
for maximum signal intensity by using the automated instrument tuning
feature while monitoring the background ion signal of m/z = 371.1 amu (decamethylcyclopentasiloxane)
to create the tune file used for data analysis. This resulted in an
S-Lens RF level setting of 62%.
onto a NanoLC-Ultra 2D HPLC (Eksigent, Dublin, CA) system equipped
with a 5 μL injection loop. Separation was performed with a
capillary column (75 μm ID, 10 cm length, 15 μm orifice)
created by hand packing a commercially available fused-silica emitter
(New Objective, Woburn MA) with 5 μm Luna C18 bonded separation
media (Phenomenex, Torrance, CA). The flow rate was 1000 nL/min for
5.5 min, then decreased to 300 nL/min with a 40 min linear gradient
of 2 to 30% CH3CN in 5 mM NH4OAc aqueous buffer
(pH 6.8), followed by a 5:95 buffer/CH3CN hold for 10 min
and a 5 min re-equilibration at 1000 nL/min 98:2 buffer/CH3CN. The injection valve was switched at 6 min to remove the sample
loop from the flow path during the gradient. Samples were analyzed
by nanoelectrospray using an LTQ Orbitrap Velos instrument (Thermo
Scientific, Waltham, MA). The nanoelectrospray source voltage was
set at 1.6 kV, and the capillary temperature was 350 °C. The
ion focusing and transfer elements of the instrument were adjusted
for maximum signal intensity by using the automated instrument tuning
feature while monitoring the background ion signal of m/z = 371.1 amu (decamethylcyclopentasiloxane)
to create the tune file used for data analysis. This resulted in an
S-Lens RF level setting of 62%.
4
Separation and Analysis of ctDNA by nanoLC-MS
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