Dataset Information

Complementary Proteome and Glycoproteome Access Revealed Through Comparative Analysis of Reversed Phase and Porous Graphitic Carbon Chromatography

ABSTRACT: Aided by improvements in instrumental and analytical techniques, rigorous connections have been drawn between glycoprotein expression, modification heterogeneity, and a variety of human illnesses. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. Instrumental advancements, optimal and nascent dissociation techniques, and computational strategies present the largest area of glycoprotein profiling innovation, often neglecting potential benefits provided by alternative chromatography techniques. Porous graphitic carbon (PGC) represents a separation regime that has gained significant interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomics experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates the advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved when in both single and biphasic PGC separation, providing a means towards facile, multiplex glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Daniel Delafield

LAB HEAD: Lingjun Li

PROVIDER: PXD024196 | Pride | 2022-02-14

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20200312_GD_PANC1_Biphasic_30C.mzXML	Mzxml
	20200312_GD_PANC1_Biphasic_30C.raw	Raw
	20200312_GD_PANC1_Biphasic_45C.mzXML	Mzxml
	20200312_GD_PANC1_Biphasic_45C.raw	Raw
	20200312_GD_PANC1_Biphasic_60C.mzXML	Mzxml

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Publications

Complementary proteome and glycoproteome access revealed through comparative analysis of reversed phase and porous graphitic carbon chromatography.

Delafield Daniel G DG Miles Hannah N HN Liu Yuan Y Ricke William A WA Li Lingjun L

Analytical and bioanalytical chemistry 20220209 18

Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alterna ...[more]

PMID: 35137243

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Project description:Understanding the response processes in cellular systems to external perturbations is a central goal of large-scale molecular profiling experiments. We investigated the molecular response of yeast to increased and lowered temperatures relative to optimal reference conditions across two levels of molecular organization: the transcriptome using a whole yeast genome microarray and the metabolome applying the GC/MS technology with in-vivo stable-isotope labeling for accurate relative quantification of a total of 50 different metabolites. The molecular adaptation of yeast to increased or lowered temperatures relative control conditions at both the metabolic and transcriptional level is dominated by temperature-inverted differential regulation patterns of transcriptional and metabolite responses and the temporal response observed to be biphasic. The set of previously described general environmental stress response (ESR) genes showed particularly strong temperature-inverted response patterns. Among the metabolites measured, trehalose was detected to respond strongest to the temperature stress and with temperature-inverted up and downregulation relative to control, mid-temperature conditions. Although associated with the same principal environmental parameter, the two different temperature regimes caused very distinct molecular response patterns at both the metabolite and the transcript level. While pairwise correlations between different transcripts and between different metabolites were found generally preserved under the various conditions, substantial differences were also observed indicative of changed underlying network architectures or modified regulatory relationships. Gene and associated gene functions were identified that are differentially regulated specifically under the gradual stress induction applied here compared to abrupt stress exposure investigated in previous studies, including genes of as of yet unidentified function and genes involved in protein synthesis and energy metabolism. Reference: Strassburg K, Walther D, Takahashi H, Kanaya S, and Kopka J. Dynamic transcriptional and metabolic responses in yeast adapting to temperature stress. OMICS JIB, 2010, 14(3), in press. Time-series with 8 time points for three different ambient temperatures. Single replicate per time point and temperature series.