Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry

Adamczyk, Maciej; Gebler, John C.; Wu, Jiang

doi:10.1002/rcm.394

Cited by 106 publications

(85 citation statements)

References 37 publications

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“…Mapping of phosphorylation sites within complex protein mixtures, however, is still a difficult task because of the often low stoichiometry of phosphorylation and signal suppression by unphosphorylated peptides. Specific capture of phosphopeptides is possible by ␤-elimination of the phosphate group and subsequent introduction of an affinity tag (18)(19)(20)(21)(22)(23), or by covalent capture and release (24). These two methods have been designed for enhanced specificity but involve complex chemistry and have not been widely used because of limited sensitivity.…”

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confidence: 99%

Phosphoproteome analysis of the human mitotic spindle

Nousiainen

Silljé

Sauer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

303

260

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During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle. mass spectrometry ͉ mitosis ͉ molecular cell biology ͉ phosphorylation ͉ proteomics A t the transition from interphase to mitosis, the microtubule network undergoes a profound change that culminates in the formation of the spindle apparatus. The mitotic spindle then serves to segregate the chromosomes to opposite poles of the cell and to define the plane of cell division. A large number of proteins associate with this microtubule-based structure and regulate its dynamic formation and function (1, 2). Several spindle proteins, including XKCM1 and XMAP215 family members (3, 4), have been identified that either stabilize or destabilize microtubules by mediating the rapid changes between polymerization and depolymerization (5, 6). These proteins play an important role in spindle formation, as illustrated by the striking change in microtubule half-life from 5-10 min in interphase to less than 1 min in mitosis (7), which results in the short and unstable microtubules characteristic of mitosis (5, 6). Another important group of spindle proteins comprises motors of the kinesin and dynein families that are essential for mitotic progression (5,6,8,9). They push the spindle poles away from each other during early mitosis and play crucial roles in capturing chromosomes and positioning them at the metaphase plate. Subsequently, motors also contribute to central spindle formation and cytokinesis. In addition, numerous structural prote...

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confidence: 99%

Phosphoproteome analysis of the human mitotic spindle

Nousiainen

Silljé

Sauer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

303

260

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“…The resulting modified peptide can then be cleaved phosphospecifically by LysC endoprotease. Other chemical derivatization approaches have been developed based on the incorporation of biotin or fluorous affinity tags to aid the enrichment of phosphopeptides [22][23][24]. Although each of these methods has its own advantages and certain limitations, the development of new approaches for unambiguous, specific, and selective analysis of phosphoproteins to reduce suppression effects and enhance the detectability of phosphopeptides is still ongoing.…”

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confidence: 99%

Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag

Jalili

Sharma

Ball

2007

J. Am. Soc. Mass Spectrom.

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To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni 2ϩ -IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot ␤-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni 2ϩ -IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per

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“…To simplify the identification of phosphoserine residues by CAD, we converted the phosphoserine residues to S-ethylcysteine by ␤-elimination in the presence of ethanethiol. This reaction specifically replaces phosphoryl group on serine or threonine with ethanethionyl giving a 44.008-Da mass shift from normal serine or threonine (17,23,24,33). Unlike phosphoryl groups, ethanethionyl groups were more resistant to fragmentation by CAD so that fragmentation of the peptide mainly takes place at the peptide backbone (17,23).…”

Section: Resultsmentioning

confidence: 99%

Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1

Uchiki

Dice

Hettich

et al. 2004

Journal of Biological Chemistry

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Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSAS-ASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser , Ser 58, and Ser 60 by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser 56 , Ser 58, and Ser 60 in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the ؉3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif.In the yeast Saccharomyces cerevisiae, Mec1 and Rad53 kinases are transducers of all known cell cycle checkpoint pathways (1). In response to DNA damage, cell cycle progression is arrested at certain phases known as checkpoints, and simultaneously cells increase their capacity to repair DNA damage. A downstream effector of the pathway modulated by Mec1 and Rad53 is ribonucleotide reductase (RNR).1 RNR catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates, which is the rate-limiting step for dNTP synthesis. Overexpression of RNR genes rescues lethality caused by deletion of MEC1 and RAD53 genes (2). During DNA damage and at S phase, levels of dNTP pools are increased to enhance the capacity of DNA repair (2). This is achieved by transcriptional activation of RNR genes in a Mec1/Rad53-dependent manner (3, 4). The activity of RNR in yeast is also regulated by the small regulatory protein Sml1 (2). Sml1 inhibits RNR activity through an interaction with the large subunit of the RNR complex, Rnr1 (2, 5). In response to DNA damage or at S phase, the intracellular level of Sml1 is reduced significantly (6), resulting in the activation of RNR.Zhao and Rothstein (7) demonstrated that the removal of ...

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Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry

Cited by 106 publications

References 37 publications

Phosphoproteome analysis of the human mitotic spindle

Phosphoproteome analysis of the human mitotic spindle

Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag

Identification of Phosphorylation Sites on the Yeast Ribonucleotide Reductase Inhibitor Sml1

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