Limits...
Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction.

Parviainen VI, Joenväärä S, Tohmola N, Renkonen R - Proteome Sci (2013)

Bottom Line: Cell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes.Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance.Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Transplantation Laboratory, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital, Helsinki, Finland. ville.parviainen@helsinki.fi.

ABSTRACT

Background: Cell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes. The surface sialylation pattern of cells is influenced by the cellular environment but also by the Golgi sialyltransferase activity and flux of metabolites through sialic acid producing pathways. Altered cell surface sialic acid patterns have been observed in several cancers and other pathological conditions. In this experiment we examined the cellular proteomic changes that occur in human embryonic kidney cells after 24 hours of sialic acid overproduction using N-Acetylmannosamine. We utilized high resolution mass spectrometry and label free protein quantification to characterize the relative changes in protein abundance as well as multiple reaction monitoring to quantify the cellular sialic acid levels.

Results: Using N-Acetylmannosamine we were able to induce sialic acid production to almost 70-fold compared to non-induced control cells. Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance. Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions. We also identified several physically interacting co-regulated proteins in the set of changed proteins.

Conclusions: In this experiment we show that increased metabolic flux through sialic acid producing pathway affects the abundance of several protein transport, epithelial adherens junction, signaling and metabolic pathway related proteins.

No MeSH data available.


Related in: MedlinePlus

Neu5Ac and ManNAc quantification with MRM. A: Neu5Ac amount in control samples without ManNAc induction. B: Neu5Ac amount in induced samples with induction using 30 mM ManNAc. C: ManNAc amount in induced samples using 30 mM ManNAc. Error bars represent lowest and highest quantification results of the three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750590&req=5

Figure 2: Neu5Ac and ManNAc quantification with MRM. A: Neu5Ac amount in control samples without ManNAc induction. B: Neu5Ac amount in induced samples with induction using 30 mM ManNAc. C: ManNAc amount in induced samples using 30 mM ManNAc. Error bars represent lowest and highest quantification results of the three biological replicates.

Mentions: The control experiments showed no detectable change in the concentration of Neu5Ac during 24 hours of measurements (Figure 2A). ManNAc- levels were too low on control samples for reliable quantification across all time points. However, the level of Neu5Ac increased extensively in the induced samples (Figure 2B). No rise was seen in the 15 and 30 minute samples but after one hour the levels began to steadily rise. After 24 hours the Neu5Ac level had increased almost 70-fold. The ManNAc level in induced samples began to increase almost immediately after induction but seemed to stabilize after one hour to an average fold increase of 2.7 (Figure 2C). After five hours the levels of ManNAc again began to grow and after 24 hours had increased to about 4.8-fold compared to the original zero hour sample.


Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction.

Parviainen VI, Joenväärä S, Tohmola N, Renkonen R - Proteome Sci (2013)

Neu5Ac and ManNAc quantification with MRM. A: Neu5Ac amount in control samples without ManNAc induction. B: Neu5Ac amount in induced samples with induction using 30 mM ManNAc. C: ManNAc amount in induced samples using 30 mM ManNAc. Error bars represent lowest and highest quantification results of the three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750590&req=5

Figure 2: Neu5Ac and ManNAc quantification with MRM. A: Neu5Ac amount in control samples without ManNAc induction. B: Neu5Ac amount in induced samples with induction using 30 mM ManNAc. C: ManNAc amount in induced samples using 30 mM ManNAc. Error bars represent lowest and highest quantification results of the three biological replicates.
Mentions: The control experiments showed no detectable change in the concentration of Neu5Ac during 24 hours of measurements (Figure 2A). ManNAc- levels were too low on control samples for reliable quantification across all time points. However, the level of Neu5Ac increased extensively in the induced samples (Figure 2B). No rise was seen in the 15 and 30 minute samples but after one hour the levels began to steadily rise. After 24 hours the Neu5Ac level had increased almost 70-fold. The ManNAc level in induced samples began to increase almost immediately after induction but seemed to stabilize after one hour to an average fold increase of 2.7 (Figure 2C). After five hours the levels of ManNAc again began to grow and after 24 hours had increased to about 4.8-fold compared to the original zero hour sample.

Bottom Line: Cell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes.Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance.Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Transplantation Laboratory, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital, Helsinki, Finland. ville.parviainen@helsinki.fi.

ABSTRACT

Background: Cell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes. The surface sialylation pattern of cells is influenced by the cellular environment but also by the Golgi sialyltransferase activity and flux of metabolites through sialic acid producing pathways. Altered cell surface sialic acid patterns have been observed in several cancers and other pathological conditions. In this experiment we examined the cellular proteomic changes that occur in human embryonic kidney cells after 24 hours of sialic acid overproduction using N-Acetylmannosamine. We utilized high resolution mass spectrometry and label free protein quantification to characterize the relative changes in protein abundance as well as multiple reaction monitoring to quantify the cellular sialic acid levels.

Results: Using N-Acetylmannosamine we were able to induce sialic acid production to almost 70-fold compared to non-induced control cells. Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance. Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions. We also identified several physically interacting co-regulated proteins in the set of changed proteins.

Conclusions: In this experiment we show that increased metabolic flux through sialic acid producing pathway affects the abundance of several protein transport, epithelial adherens junction, signaling and metabolic pathway related proteins.

No MeSH data available.


Related in: MedlinePlus