High-resolution peptide separations using nano-LC at ultra-high pressure
We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The s...
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Published in | Journal of separation science Vol. 36; no. 7; pp. 1192 - 1199 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Weinheim
Blackwell Publishing Ltd
01.04.2013
Wiley Wiley Subscription Services, Inc |
Subjects | |
Online Access | Get full text |
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Abstract | We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions. |
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AbstractList | We report on the optimization of nano‐
LC
gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica
C
18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome
c
tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an
Escherichia
coli
digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (
RSD
values below 0.18%) was demonstrated applying ultra‐high pressure conditions. We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 mu m id capillary columns packed with 2, 3, and 5 mu m fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 mu m particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2- mu m packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions. We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 µm id capillary columns packed with 2, 3, and 5 µm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 µm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-µm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions. [PUBLICATION ABSTRACT] We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions. |
Author | Desmet, Gert Eeltink, Sebastiaan Nováková, Lucie De Pra, Mauro Vaast, Axel Stassen, Catherine Swart, Remco Broeckhoven, Ken |
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CitedBy_id | crossref_primary_10_1016_j_aca_2014_09_006 crossref_primary_10_1016_j_chroma_2022_463251 crossref_primary_10_1002_jssc_201600775 crossref_primary_10_1016_j_aca_2019_08_068 crossref_primary_10_1016_j_chroma_2015_07_090 crossref_primary_10_1002_jssc_202100116 crossref_primary_10_1016_j_jprot_2017_06_013 crossref_primary_10_1007_s10337_018_3647_5 crossref_primary_10_4155_fsoa_2016_0014 crossref_primary_10_1002_jssc_201500765 crossref_primary_10_4155_bio_15_92 crossref_primary_10_56530_lcgc_eu_bm1469g6 crossref_primary_10_1016_j_chroma_2021_462310 crossref_primary_10_1021_acs_analchem_5b04093 |
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Keywords | Capillary column Ultra-high pressures Elution Gradient Peak resolution Chemical analysis Flow rate Nano flow rate Peptides HPLC chromatography Kinetic method Kinetic plot Packed capillary columns Protein Operating conditions Column coupling Reversed phase chromatography Ultraviolet detector Pressure effect Analysis method Plate efficiency Peptide fragment Proteomics Packed column |
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Snippet | We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by... We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by... We report on the optimization of nano‐ LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by... |
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SubjectTerms | Analytical, structural and metabolic biochemistry Biological and medical sciences Capillarity Chromatography Chromatography, High Pressure Liquid Column coupling Column packings Fundamental and applied biological sciences. Psychology General aspects, investigation methods Kinetic plot Nano flow rate Nanomaterials Nanostructure Nanotechnology - methods Optimization Packed capillary columns Particle Size Peptides Peptides - chemistry Peptides - isolation & purification Pressure Proteins Proteomics Separation Steepness Ultra-high pressures |
Title | High-resolution peptide separations using nano-LC at ultra-high pressure |
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