Direct Analysis of the Products of Sequential Cleavages of Peptides and Proteins Affinity-Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Qian, Xiaohong; Zhou, Wei; Khaledi, Morteza G.; Tomer, Kenneth B.

doi:10.1006/abio.1999.4268

Cited by 21 publications

(15 citation statements)

References 24 publications

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“…Earlier, Tomer and co-workers have reported that direct MALDI could account for the metal-binding peptide signals observed in MALDI-TOFMS spectra obtained by loading IMAC beads onto a MALDI target in CHCA [9,10]. These studies were recently extended to the use of Fe 3ϩ -and Ga 3ϩ -immobilized beads for the selective detection of phosphopeptides [7].…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…Mass spectrometry (MS), combined with separation methods such as PAGE and high-performance liquid chromatography (HPLC), has emerged as a key technology for studying the spatial and temporal expression of proteins [13]. Mass analysis is usually performed on peptides obtained by tryptic digestion of proteins following gel electrophoresis, LC or affinity purification [9,12,[13][14][15][16][17][18][19][20][21][22][23][24]. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is a powerful method for peptide sequencing and protein identification, especially when combined with capillary electrophoresis or HPLC separation, automated MS/MS and database search procedures [13,18,21,[25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Ross

Lee

Smith³

et al. 2002

Proteomics

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Protein identification by peptide mass mapping usually involves digestion of gel-separated proteins with trypsin, followed by mass measurement of the resulting peptides by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Positive identification requires measurement of enough peptide masses to obtain a definitive match with sequence information recorded in protein or DNA sequence databases. However, competitive binding and ionization of residual surfactant introduced during polyacrylamide gel electrophoresis (PAGE) can inhibit solid-phase extraction and MS analysis of tryptic peptides. We have evaluated a novel, acid-labile surfactant (ALS) as an alternative to sodium dodecylsulfate (SDS) for two-dimensional (2-D) PAGE separation and MALDI-MS mapping of proteins. ALS was substituted for SDS at the same concentration in buffers and gels used for 2-D PAGE. Manual and automated procedures for spot cutting and in-gel digestion were used to process Coomassie stained proteins for MS analysis. Results indicate that substituting ALS for SDS during PAGE can significantly increase the number of peptides detected by MALDI-MS, especially for proteins of relatively low abundance. This effect is attributed to decomposition of ALS under acidic conditions during gel staining, destaining, peptide extraction and MS sample preparation. Automated excision and digestion procedures reduce contamination by keratin and other impurities, further enhancing MS identification of gel separated proteins.

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“…The initial experiments with attached affinity ligands on beads for direct MALDI desorption were described under the term surface-enhanced affinity capture (SEAC) [93][94][95]. Alternatively, affinity ligands can be immobilized on the MALDI probe via a gold foil [96] or a nitrocellulose film [97].…”

Section: Affinity Interactions In Msmentioning

confidence: 99%

Immobilized microfluidic enzymatic reactors

2004

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The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology.

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Direct Analysis of the Products of Sequential Cleavages of Peptides and Proteins Affinity-Bound to Immobilized Metal Ion Beads by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Cited by 21 publications

References 24 publications

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

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

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

Immobilized microfluidic enzymatic reactors

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