Rapid Enrichment of Phosphopeptides from Tryptic Digests of Proteins Using Iron Oxide Nanocomposites of Magnetic Particles Coated with Zirconia as the Concentrating Probes

Abstract

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is approximately 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipetting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.

Microwave Assisted Reactions in Organic Chemistry: A Review of Recent Advances

“…If we load digests with magnetic elements to enhance the microwave absorptivity of the irradiated solutions, similar improvements in terms of digest efficiency/sequence coverage could be observed (Y. Lo et al, 2007;Li et al, 2007).…”

The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis

“…Incubation time (min) detecting phosphopeptides, which is lower than that of other approaches by using TiO 2 beads, ZrO 2 beads and Fe 3ϩ -immobilized affinity chromatography as the affinity probes in conjunction with MALDI-TOF MS analysis [15,17,18,31]. The lower limit of detection for phosphopeptide could be attributed to large surface-tovolume ratio of the magnetic nanoparticle, which results in the high loading capacity of Zr 4ϩ cations and consequently large trapping capacity towards phosphopeptides of proteolytic digests.…”

“…

The© highly© selective© capture© of© phosphopeptides© from© proteolytic© digests© is© a© great© challenge for© the© identification© of© phosphoproteins© by© mass© spectrometry.© In© this© work,© the© zirconium phosphonate-modified© magnetic© Fe 3 O 4 /SiO 2© core/shell© nanoparticles© have© been© synthesized and© successfully© applied© for© the© selective© capture© of© phosphopeptides© from© complex© tryptic digests© of© proteins© before© the© analysis© of© MALDI-TOF© mass© spectrometry© with© the© desired convenience©of©sample©handling.©The©ratio©of©magnetic©nanoparticle©to©protein©and©the incubation© time© for© capturing© phosphopeptides© from© complex© proteolytic© digests© were© investigated,© and© the© optimized© nanoparticle-to-protein© ratio© and© incubation© time© were© between© 15:1© to 30:1© and© 30© min,© respectively.© The© excellent© detection© limit© of© 0.5© fmol© ␤-casein© has© been© achieved by© MALDI-TOF© mass© spectrometry© with© the© specific© capture© of© zirconium© phosphonate-modified magnetic©Fe 3 O 4 nanoparticles.©The©great©specificity©of©zirconium©phosphonate-modified magnetic© Fe 3 O 4© nanoparticles© to© phosphopeptides© was© demonstrated© by© the© selective© capture of© phosphopeptides© from© a© complex© tryptic© digest© of© the© mixture© of© ␣-casein© and© bovine© serum albumin© at© molar© ratio© of© 1© to© 100© in© MALDI-TOF-MS© analysis.© An© application© of© the© magnetic nanoparticles© to© selective© capture© phosphopeptides© from© a© tryptic© digest© of© mouse© liver© lysate was© further© carried© out© by© combining© with© nano-LC-MS/MS© and© MS/MS/MS© analyses,© and .©The©comprehensive©understanding©of©biological processes© requires© the© characterization© of© protein© phosphorylation© at© molecular© level.© Mass© spectrometry© is© a fundamental© tool© for© detecting© and© mapping© the© phosphoryl© modifications© of© proteins© [2][3][4][5].© However,© due© to the© minimal© amount© of© phosphorylation© found© on© proteins©and©the©serious©ion©suppression©phenomenon resulting© from© the© proteolytic© complexity© [6],© selective capture© of© phosphopeptides© from© complex© proteolytic products© has© been© remaining© quite© a© challenge© in© the field© of© proteomics.Up© to© now,© a© variety© of© approaches© have© been© developed© for© selective© determination© of© phosphopeptides© and thus© improve© the© detection© sensitivity© of© mass© spectrometry,© such© as© chemical© derivatization© [7][8][9],© immunoprecipitation© [10,© 11],© strong© ion-exchange© [12,© 13],© and© metal affinity© technique© [14© -24].© Among© these,© the© metal© affinity technique© using© immobilized© metal© ions© or© metal© oxides as© the© affinity© moieties© to© phosphopeptides© was© the© most frequently© applied© method,© and© has© been© widely© applied for©the©selective©capture©or©even©purification©of©phos-phopeptides© via© the© specific© binding© between© the© metal moieties©and©the©phosphoryl©groups©of©peptides©and proteins.© For© instance,© the© immobilized© metal© ions© of© Fe 3ϩ , Ga 3ϩ ,© and© Zr 4ϩ© on© chromatographic© adsorbents© showed the©specific©binding©characteristics©to©capture©phos-phopeptides© [20,© 23,© 24],© and© the© titanium© dioxide© (TiO 2 ) and© zirconium© dioxide© (ZrO 2 )© demonstrated© the© binding specificity© to© phosphopeptides© of© the© complex© proteolytic digests© via© the© bidentate© interactions© [17][18]

…”

“…Up© to© now,© a© variety© of© approaches© have© been© developed© for© selective© determination© of© phosphopeptides© and thus© improve© the© detection© sensitivity© of© mass© spectrometry,© such© as© chemical© derivatization© [7][8][9],© immunoprecipitation© [10,© 11],© strong© ion-exchange© [12,© 13],© and© metal affinity© technique© [14© -24].© Among© these,© the© metal© affinity technique© using© immobilized© metal© ions© or© metal© oxides as© the© affinity© moieties© to© phosphopeptides© was© the© most frequently© applied© method,© and© has© been© widely© applied for©the©selective©capture©or©even©purification©of©phos-phopeptides© via© the© specific© binding© between© the© metal moieties©and©the©phosphoryl©groups©of©peptides©and proteins.© For© instance,© the© immobilized© metal© ions© of© Fe 3ϩ , Ga 3ϩ ,© and© Zr 4ϩ© on© chromatographic© adsorbents© showed the©specific©binding©characteristics©to©capture©phos-phopeptides© [20,© 23,© 24],© and© the© titanium© dioxide© (TiO 2 ) and© zirconium© dioxide© (ZrO 2 )© demonstrated© the© binding specificity© to© phosphopeptides© of© the© complex© proteolytic digests© via© the© bidentate© interactions© [17][18][19].…”

“…For example, Chen et al [17,18] used the TiO 2 and ZrO 2 -coated iron oxide magnetic nanoparticles as the effective affinity probes for the selective concentration of phosphopeptides from the tryptic protein digests in MALDI-TOF MS with a low-femtomole limit of detection; Tan et al [30] directly employed the TiO 2 -modified magnetic nanoparticles on MALDI targets, which was also demonstrated the specific capture and confident detection of phosphopeptides in MALDI-TOF-MS.…”