Limits...
Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography.

Hao P, Guo T, Sze SK - PLoS ONE (2011)

Bottom Line: Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue.In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein.Interestingly, partially modified proteins tend to occur on proteins involved in transport.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

Show MeSH

Related in: MedlinePlus

The occurrence of partial phosphorylation of a representative peptide.The ERLIC chromatogram shows the fractions in which the phosphopeptide and its unmodified form were eluted (A); Representative MS/MS spectra for identification of the phosphopeptide (B) and its unmodified form (C); Gene ontology analysis of partially phosphorylated proteins according to their subcellular locations (D) and biological processes (E) using AmiGo Go Slimmer.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044146&req=5

pone-0016884-g004: The occurrence of partial phosphorylation of a representative peptide.The ERLIC chromatogram shows the fractions in which the phosphopeptide and its unmodified form were eluted (A); Representative MS/MS spectra for identification of the phosphopeptide (B) and its unmodified form (C); Gene ontology analysis of partially phosphorylated proteins according to their subcellular locations (D) and biological processes (E) using AmiGo Go Slimmer.

Mentions: Protein phosphorylation can vary quickly in response to intracellular or extracellular stimuli independent of protein expression, and a change in phosphorylation level of some proteins may be an indicator of physiological state [19]. Sometimes a change in protein phosphorylation also accompanies a protein's expression [42]. Thus, it is necessary to distinguish between the two. The presented method (ERLIC04) for concurrent analysis of proteome and subproteomes is a promising means of doing so. By comparing phosphopeptides with unmodified peptides identified here, the corresponding unmodified peptides were found for 96 unique phosphopeptides in 84 phosphoproteins (Table S5). As shown in Figure 4A, the phosphopeptide of LCLpSTVDLEVK was eluted 11 fractions later than its unmodified form, due of course to the phosphate group that prolongs its retention in the ERLIC mode. The phosphorylation level of the protein at this site was estimated to be about 51% using the peak intensities of the extracted ion chromatogram (XIC) of the phosphopeptide and its unmodified form. This estimate presumes that they ionize with equal efficiency, which may or may not be the case.


Simultaneous analysis of proteome, phospho- and glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction chromatography.

Hao P, Guo T, Sze SK - PLoS ONE (2011)

The occurrence of partial phosphorylation of a representative peptide.The ERLIC chromatogram shows the fractions in which the phosphopeptide and its unmodified form were eluted (A); Representative MS/MS spectra for identification of the phosphopeptide (B) and its unmodified form (C); Gene ontology analysis of partially phosphorylated proteins according to their subcellular locations (D) and biological processes (E) using AmiGo Go Slimmer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044146&req=5

pone-0016884-g004: The occurrence of partial phosphorylation of a representative peptide.The ERLIC chromatogram shows the fractions in which the phosphopeptide and its unmodified form were eluted (A); Representative MS/MS spectra for identification of the phosphopeptide (B) and its unmodified form (C); Gene ontology analysis of partially phosphorylated proteins according to their subcellular locations (D) and biological processes (E) using AmiGo Go Slimmer.
Mentions: Protein phosphorylation can vary quickly in response to intracellular or extracellular stimuli independent of protein expression, and a change in phosphorylation level of some proteins may be an indicator of physiological state [19]. Sometimes a change in protein phosphorylation also accompanies a protein's expression [42]. Thus, it is necessary to distinguish between the two. The presented method (ERLIC04) for concurrent analysis of proteome and subproteomes is a promising means of doing so. By comparing phosphopeptides with unmodified peptides identified here, the corresponding unmodified peptides were found for 96 unique phosphopeptides in 84 phosphoproteins (Table S5). As shown in Figure 4A, the phosphopeptide of LCLpSTVDLEVK was eluted 11 fractions later than its unmodified form, due of course to the phosphate group that prolongs its retention in the ERLIC mode. The phosphorylation level of the protein at this site was estimated to be about 51% using the peak intensities of the extracted ion chromatogram (XIC) of the phosphopeptide and its unmodified form. This estimate presumes that they ionize with equal efficiency, which may or may not be the case.

Bottom Line: Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue.In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein.Interestingly, partially modified proteins tend to occur on proteins involved in transport.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.

ABSTRACT
Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

Show MeSH
Related in: MedlinePlus