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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.

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Related in: MedlinePlus

Comparison of the efficiency of SCX and ERLIC methods.Comparison of total protein (A), phosphoprotein (B) and glycoprotein (C) identification in SCX and ERLIC methods using different gradients. For the gradients used in each separation method, see Materials and Methods.
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pone-0016884-g002: Comparison of the efficiency of SCX and ERLIC methods.Comparison of total protein (A), phosphoprotein (B) and glycoprotein (C) identification in SCX and ERLIC methods using different gradients. For the gradients used in each separation method, see Materials and Methods.

Mentions: As shown in Figure 2A, the number of proteins identified in ERLIC04 was the highest in all six fractionation methods, i.e. 2929, better than with the SCX method that is so widely used for the fractionation of unmodified peptides. As illustrated in Figure 2B, 338 phosphoproteins and 583 phosphorylation sites (The MS/MS spectra are shown in Data S1) were identified in ERLIC04, the highest of the six fractionation methods (158 and 204 percent higher, respectively, than with SCX01 in which 131 phosphoproteins and 192 phosphorylation sites were identified). The identification of significantly less phosphoproteins in SCX may be attributed to 1) the loss of some hydrophilic phosphopeptides during the desalting step and 2) the distribution of most phosphopeptides amongst coeluting unmodified peptides. The better separation and enrichment efficiency of ERLIC can be another important factor. The number of glycoproteins (387) and glycosylation sites (722) (The MS/MS spectra are shown in Data S2) identified in ERLIC04 was also the highest compared with the other fractionation methods (Figure 2C). The MS/MS spectra for the identification of phosphopeptides and glycopeptides were shown in Data S3–S4. For SCX01, 598 glycosylation sites in 353 glycoproteins were identified. In conclusion, ERLIC04 using 80% ACN, 0.1% FA as buffer A and 10% ACN, 2% FA as buffer B performed the best in the concurrent fractionation of unmodified peptides, phosphopeptides and glycopeptides. The complete list of proteins, phosphoproteins and glycoproteins identified in this study is supplied in Table S1–S3. Use of salt-containing mobile phases (e.g., ERLIC01 and ERLIC02) led to a better separation of modified from unmodified peptides but not to the identification of more modified peptides. A possible explanation is that the distribution of phosphopeptides or glycopeptides into more fractions in the salt-free ERLIC gradient facilitates their MS analysis.


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)

Comparison of the efficiency of SCX and ERLIC methods.Comparison of total protein (A), phosphoprotein (B) and glycoprotein (C) identification in SCX and ERLIC methods using different gradients. For the gradients used in each separation method, see Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016884-g002: Comparison of the efficiency of SCX and ERLIC methods.Comparison of total protein (A), phosphoprotein (B) and glycoprotein (C) identification in SCX and ERLIC methods using different gradients. For the gradients used in each separation method, see Materials and Methods.
Mentions: As shown in Figure 2A, the number of proteins identified in ERLIC04 was the highest in all six fractionation methods, i.e. 2929, better than with the SCX method that is so widely used for the fractionation of unmodified peptides. As illustrated in Figure 2B, 338 phosphoproteins and 583 phosphorylation sites (The MS/MS spectra are shown in Data S1) were identified in ERLIC04, the highest of the six fractionation methods (158 and 204 percent higher, respectively, than with SCX01 in which 131 phosphoproteins and 192 phosphorylation sites were identified). The identification of significantly less phosphoproteins in SCX may be attributed to 1) the loss of some hydrophilic phosphopeptides during the desalting step and 2) the distribution of most phosphopeptides amongst coeluting unmodified peptides. The better separation and enrichment efficiency of ERLIC can be another important factor. The number of glycoproteins (387) and glycosylation sites (722) (The MS/MS spectra are shown in Data S2) identified in ERLIC04 was also the highest compared with the other fractionation methods (Figure 2C). The MS/MS spectra for the identification of phosphopeptides and glycopeptides were shown in Data S3–S4. For SCX01, 598 glycosylation sites in 353 glycoproteins were identified. In conclusion, ERLIC04 using 80% ACN, 0.1% FA as buffer A and 10% ACN, 2% FA as buffer B performed the best in the concurrent fractionation of unmodified peptides, phosphopeptides and glycopeptides. The complete list of proteins, phosphoproteins and glycoproteins identified in this study is supplied in Table S1–S3. Use of salt-containing mobile phases (e.g., ERLIC01 and ERLIC02) led to a better separation of modified from unmodified peptides but not to the identification of more modified peptides. A possible explanation is that the distribution of phosphopeptides or glycopeptides into more fractions in the salt-free ERLIC gradient facilitates their MS analysis.

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