Analytical methodologies for the detection and structural characterization of phosphorylated proteins

D’Ambrosio, Chiara; Salzano, Anna Maria; Arena, Simona; Renzone, Giovanni; Scaloni, Andrea

doi:10.1016/j.jchromb.2006.06.033

Cited by 28 publications

(14 citation statements)

References 189 publications

(310 reference statements)

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“…Because recent reviews focused on IMAC and covalent capture (Reinders & Sickmann, 2005; Morandell et al, 2006; Schmelzle & White, 2006; D'Ambrosio et al, 2007; Yu, Issaq, & Veenstra, 2007), we will present these methods relatively briefly and will describe advances in MOAC in more detail. Section III of this review describes the adaptation of IMAC and MOAC for selective capture on magnetic beads and isolation of phosphopeptides directly on MALDI plates.…”

Section: Introductionmentioning

confidence: 99%

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Dunn

Reid

Bruening

2009

Mass Spectrometry Reviews

197

192

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Mass spectrometry is the tool of choice to investigate protein phosphorylation, which plays a vital role in cell regulation and diseases such as cancer. However, low abundances of phosphopeptides and low degrees of phosphorylation typically necessitate isolation and concentration of phosphopeptides prior to MS analysis. This review discusses the enrichment of phosphopeptides with immobilized metal affinity chromatography, reversible covalent binding, and metal oxide affinity chromatography. Capture of phosphopeptides on TiO(2) seems especially promising in terms of selectivity and recovery, but the success of all methods depends on careful selection of binding, washing, and elution solutions. Enrichment techniques are complementary, such that a combination of methods greatly enhances the number of phosphopeptides isolated from complex samples. Development of a standard series of phosphopeptides in a highly complex mixture of digested proteins would greatly aid the comparison of different enrichment methods. Phosphopeptide binding to magnetic beads and on-plate isolation prior to MALDI-MS are emerging as convenient methods for purification of small (microL) samples. On-plate enrichment can yield >70% recoveries of phosphopeptides in mixtures of a few digested proteins and can avoid sample-handling steps, but this technique is likely limited to relatively simple samples such as immunoprecipitates. With recent advances in enrichment techniques in hand, MS analysis should provide important insights into phosphorylation pathways.

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Section: Introductionmentioning

confidence: 99%

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Dunn

Reid

Bruening

2009

Mass Spectrometry Reviews

197

192

View full text Add to dashboard Cite

show abstract

“…Recent years have shown a notable increase in the number of analytical tools available to study phosphorylation events on a large scale [1][2][3][4][5]. These methods for "phosphoproteomics" have complemented previously available tools such as phosphospecific antibodies and immobilized metal affinity chromatography (IMAC) and include methods based on metal oxide affinity chromatography (MOAC, see below), ion exchange chromatography [6][7][8][9] as well as methods involving chemical modification reactions [10,11].…”

Section: Introductionmentioning

confidence: 99%

Optimizing the performance of tin dioxide microspheres for phosphopeptide enrichment

Leitner¹,

Sturm²,

Smått³

et al. 2009

Analytica Chimica Acta

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“…We note that multiply phosphorylated peptides may be more difficult to detect as positive ions because of their low basicity and therefore lower ionization efficiency in electrospray 22. The same problem is encountered with phosphopeptide ions produced by protonation 23, 24. Our continuing studies on optimization of ionization conditions will address this issue with tryptic digests from in vitro protein phosphorylation.…”

Section: Resultsmentioning

confidence: 96%

Gallium metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides in complex mixtures

Blacken¹,

Sadı́lek²,

Tureček³

2008

J. Mass Spectrom.

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Metal affinity capture tandem mass spectrometry (MAC-MSMS) is evaluated in a comparative study of a lysine-derived nitrilotriacetic acid (Nα, Nα-bis-(carboxymethyl)lysine, LysNTA) and an aspartic-acid-related iminodiacetic acid (N-(4-aminobutyl)aspartic acid, AspIDA) as selective phosphopeptide detection reagents. Both LysNTA and AspIDA spontaneously form ternary complexes with GaIII and phosphorylated amino acids and phosphopeptides upon mixing in solution. Collision-induced dissociation of positive complex ions produced by electrospray produces common fragments (LysNTA + H)+ or (AspIDA + H)+ at m/z 263 and 205, respectively. MSMS precursor scans using these fragments as reporter ions allow one to selectively detect multiple charge states of phosphopeptides in mixtures. It follows from this comparative study that LysNTA is superior to AspIDA in detecting phosphopeptides, possibly because of the higher coordination number and greater stability constant for GaIII – phosphopeptide complexation of the former reagent. In a continuing development of MAC-MSMS for proteomics applications, we demonstrate its utility in a post-column reaction format. Using a simple post-column-reaction ‘T’ and syringe pump to deliver our chelating reagents, α-casein tryptic phosphopeptides can be selectively analyzed from a solution containing a twofold molar excess of bovine serum albumin. The MAC-MSMS method is shown to be superior to the commonly used neutral loss scan for the common loss of phosphoric acid.

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Analytical methodologies for the detection and structural characterization of phosphorylated proteins

Cited by 28 publications

References 189 publications

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry

Optimizing the performance of tin dioxide microspheres for phosphopeptide enrichment

Gallium metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides in complex mixtures

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