Direct Analysis of Affinity-Bound Analytes by MALDI/TOF MS

Papac, Damon I.; Hoyes, John; Tomer, Kenneth B.

doi:10.1021/ac00089a004

Cited by 75 publications

(79 citation statements)

References 11 publications

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“…The work presented here extends the direct MALDI-MS/MS approach to sequence epitope-containing peptides bound to a variety of antibody beads. In contrast to earlier reports on MALDI-TOF instruments [4,15], no loss of mass accuracy results from analyzing the affinity beads directly on the target. The data presented in this paper demonstrate that based on the high mass accuracy obtained with a QqTOF, database searching, and de novo sequencing for protein identification can be successfully performed on the MS/MS spectra obtained, even with only femtomoles of peptide present.…”

contrasting

confidence: 59%

“…As can be seen in Figure 1a, with a maximum of 234 fmol of affinity-bound peptide loaded on the target, the protonated molecule ([M ϩ H] ϩ ) at m/z 1702.553 of the phosphorylated peptide was observed, with a signal to noise ratio (S/N) greater than 10, as well as a loss of 98 Da (m/z 1604.477), corresponding to loss of H 3 PO 4 , of approximately 25% relative intensity. The resolution obtained for the peak at m/z 1702.553 was 11,601.…”

Section: Phosphotyrosine/anti-phosphotyrosine Bindingmentioning

confidence: 99%

“…There has been some speculation in the literature whether or not the peptides are still bound to the beads following the addition of matrix solution [3,4]. Quite recently, it was reported that the DHB matrix solvent actually can "elute" the phosphorylated peptides from IMAC beads during the spotting process [3].…”

Section: Are the Peptides Bound To The Antibody Beads?mentioning

confidence: 99%

“…To obtain sequence information on a particular affinity-bound peptide, enzymatic sequencing experiments had to be performed while the peptides were still bound to the beads [4,7,8]. While this has proven to be a useful approach for epitope mapping and other applications [6, 8, 9 -11], the resulting data can be difficult to interpret if more than one peptide is isolated, or if several residues are cleaved simultaneously.…”

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confidence: 99%

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Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

Raska

Parker

Sunnarborg

et al. 2003

J. Am. Soc. Mass Spectrom.

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A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor

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contrasting

confidence: 59%

Section: Phosphotyrosine/anti-phosphotyrosine Bindingmentioning

confidence: 99%

Section: Are the Peptides Bound To The Antibody Beads?mentioning

confidence: 99%

mentioning

confidence: 99%

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Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

Raska

Parker

Sunnarborg

et al. 2003

J. Am. Soc. Mass Spectrom.

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“…Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono-and all poly-phosphopeptide species from I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].In addition to the usual conditions documented above for solubilization of peptides bound in their immobilized, chelated form attached to the resin, evidence has been presented which suggests that monophosphopeptides may be released directly from the resin during the MALDI process [7,9]. It was recognized that the ferric IMAC resin must bind multiple phosphorylated components as well, but that laser irradiation does not easily dissociate these components from the agarose IMAC beads [7].…”

mentioning

confidence: 99%

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

Hart

Waterfield

Burlingame

et al. 2002

J. Am. Soc. Mass Spectrom.

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We have revisited the direct analysis experiments reported by Tomer and co-workers in the MALDI-TOFMS analysis of phosphopeptide-loaded immobilized metal ion affinity chromatography (IMAC) beads (Zhou, W.; Merrick, B. A.; Khaledi, M. G.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 2000, 11, 273-282). The results described herein provide no evidence to support a laser-induced direct desorption of phosphopeptides chelated on IMAC beads. However, we have established that solubilization of mono-phosphopeptides from their immobilized Fe 3ϩ -NTA chelates does occur effectively in solutions containing certain MALDI matrices. Particularly effective is 2,5-dihydroxybenzoic acid (2,5-DHB), which apparently forms a stronger chelation complex with Fe 3ϩ -NTA than mono-phosphopeptides. With regard to the disparity observed between the low pH value of MALDI matrices (saturated 2,5-DHB aq ϳ pH 2) and the high pH values of conventional IMAC eluents (typically above pH 7), we have also investigated the influence of eluent pH on the recovery of phosphopeptides from IMAC media. Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono-and all poly-phosphopeptide species from I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].In addition to the usual conditions documented above for solubilization of peptides bound in their immobilized, chelated form attached to the resin, evidence has been presented which suggests that monophosphopeptides may be released directly from the resin during the MALDI process [7,9]. It was recognized that the ferric IMAC resin must bind multiple phosphorylated components as well, but that laser irradiation does not easily dissociate these components from the agarose IMAC beads [7].While it was suggested that laser desorption directly from IMAC beads occurs particularly for mono-phosphorylated peptide components, in these reports it was also questioned whether or not the IMAC analytes are still bound to the beads through their affinity interaction immediately prior to laser desorption [9,10]. Therefore, the possibility exists that "elution" from the beads into the MALDI matrix solution/crystals has in fact taken place prior to MALDI desorption and mass analysis.Of course, there would be clear advantages in having a protocol that permits the direct analysis of phosphopeptides from IMAC beads, particularly if the analyte species thus detected could be qualitatively and quantitatively representative of the components bound to the resin. However, if phosphopeptide desorption takes place directly fro...

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