Dataset Information

Improved reversed phase chromatography of hydrophilic peptides from spatial and temporal changes in column temperature

ABSTRACT: Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than -2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the co-elution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: Clifford Young

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD006112 | Pride | 2017-04-10

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20140919_QE6_LC5_CY_SA_IsoAsp_40min_12mix_01.raw	Raw
	20140919_QE6_LC5_CY_SA_IsoAsp_40min_12mix_02.raw	Raw
	20140919_QE6_LC5_CY_SA_IsoAsp_40min_12mix_05.raw	Raw
	20140919_QE6_LC5_CY_SA_IsoAsp_40min_12mix_08.raw	Raw
	20140919_QE6_LC5_CY_SA_IsoAsp_40min_12mix_09.raw	Raw

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Publications

Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature.

Young Clifford C Podtelejnikov Alexandre V AV Nielsen Michael L ML

Journal of proteome research 20170407 6

Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization-mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a two-column setup was evaluated for improvements in chromatography. Using ...[more]

PMID: 28387123

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