Phosphopeptide enrichment by IEF

Maccarrone, Giuseppina; Kolb, Nikolaus; Teplytska, Larysa; Birg, Isabel; Zollinger, Richard K.; Holsboer, Florian; Turck, Christoph W.

doi:10.1002/elps.200600145

Cited by 19 publications

(22 citation statements)

References 17 publications

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“…fractionated mixtures of three methyl-esterified synthetic peptides in different phosphorylation statuses on strips with pH range 3–8, demonstrating that phosphorylated peptides can be separated from unmodified peptides based on isoelectric point 31 . In another work, peptides derived from whole cell lysates were focused on an IPG strip ranging from pH 3 to 6, identifying 58 phospho-peptides overall 32 . Here we used a broad pH range (pH 2.5–3.7 and pH 3–10) separated into narrow pH fractions to analyze the distribution of phospho-peptides demonstrating at a high resolution a distinct fractionation pattern according to the number of phosphorylations (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Panizza

Branca

Oliviusson³

et al. 2017

Sci Rep

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Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5–3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

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Section: Discussionmentioning

confidence: 99%

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Panizza

Branca

Oliviusson³

et al. 2017

Sci Rep

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show abstract

“…Finally, IEF is a separation principle orthogonal to chromatographic procedures in general, so that it is suited for combination with a variety of other techniques for efficient separation of complex peptide mixtures. Even a phosphopeptide enrichment without methyl esterification will be useful to obtain a less complex and phosphopeptide-enriched peptide mixture [22]. Current drawbacks of the presented IEF procedure are the elaborate combination with other techniques such as MS and the occurrence of hydrolytic side reactions.…”

Section: Summary and Perspectivementioning

confidence: 99%

pI‐based phosphopeptide enrichment combined with nanoESI‐MS

2007

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IEF is introduced as a new principle for enrichment and separation of phosphopeptides as obtained after digestion of phosphoproteins by trypsin. Tryptic peptides and phosphopeptides exhibit pI values, which overlap in the range of about 4-6. However, after methyl esterification of all carboxyl functions, the pI values of tryptic peptides and phosphopeptides regroup in discrete clusters. In addition, mono- and diphosphorylated peptides show different but very homogeneous pI values, with variations when internal Arg, Lys, or His residues are present. Experimentally, this new concept was applied for separation of model peptides on IPG strips pH 3-10 as used in the first dimension of 2-DE. After IEF of methyl-esterified peptides, the IPG strip was cut into pieces followed by peptide extraction, desalting and MS analysis by nanoESI-MS. Phosphopeptides were found to focus in good agreement with their calculated pI values. This analytical strategy showed a resolution of about 0.2 pI units, and thus turned out to be capable of detecting minor differences in pI values, such as those occurring between pSer, pThr and pTyr residues. Using IPG strips with a pI range of 3-10, methyl esterified nonphosphorylated tryptic peptides are concentrated in the basic part of the IPG strip or even leave the strip. Thus, efficient enrichment of phosphopeptides and their subfractionation according to pI is obtained in one step. Minor hydrolytic side reactions including deamidation of Asn and partial hydrolysis of methyl esters are observed. The results show that IEF opens attractive avenues for the further advancement of analytical phosphoproteomics.

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“…However, the complexity and dynamics of biological samples, in particular the heterogeneity in the various magnitudes of phosphorylation signals displayed in the samples, remain as insurmountable technical difficulties. A number of approaches have been developed to address the issues relating to sample fractionation and enrichment procedures, such as peptide isoelectric focusing (34), IMAC (35), and TiO 2 affinity pulldown (23).…”

Section: Table IV Phosphopeptides Of 20 S Proteasome From Murine Livementioning

confidence: 99%

Revealing the Dynamics of the 20 S Proteasome Phosphoproteome

Zong

Wang

et al. 2008

Molecular & Cellular Proteomics

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The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO 2 ), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential ␤5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (␤1 caspase-like, ␤2 trypsin-like, and ␤5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.

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Phosphopeptide enrichment by IEF

Cited by 19 publications

References 17 publications

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

pI‐based phosphopeptide enrichment combined with nanoESI‐MS

Revealing the Dynamics of the 20 S Proteasome Phosphoproteome

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