Investigating quantitation of phosphorylation using MALDI‐TOF mass spectrometry

Parker, Laurie L.; Engel-Hall, Aaron; Drew, Kevin; Steinhardt, George F.; Helseth, Donald L.; Jabon, David; McMurry, Timothy L.; Angulo, David; Kron, Stephen J.

doi:10.1002/jms.1342

Cited by 17 publications

(21 citation statements)

References 15 publications

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“…For MALDI-TOF MS analysis, peptide was captured from cell lysates (after UV treatment to release the ‘reporter’ segment) in a 96-well plate format using streptavidin conjugated to superparamagnetic nanoparticles and analyzed in both positive and negative linear modes with MALDI-TOF MS. As commonly seen for MALDI-TOF MS and previously observed with similar peptides, the phosphopeptide ionized preferentially in negative mode. (19) Signal from unphosphorylated peptide (EAIYAAPFAKKK b G) was used as an internal standard and relative conversion was estimated by quantifying the peak integrations for the unphosphorylated and phosphorylated species from multiple experimental replicates, as described by us previously. (19)…”

Section: Resultsmentioning

confidence: 99%

“…(19) Signal from unphosphorylated peptide (EAIYAAPFAKKK b G) was used as an internal standard and relative conversion was estimated by quantifying the peak integrations for the unphosphorylated and phosphorylated species from multiple experimental replicates, as described by us previously. (19)…”

Section: Resultsmentioning

confidence: 99%

“…Samples were analyzed using a Voyager 4800 MALDI-TOF/TOF (Applied Biosystems) in linear positive and negative modes and the relative levels of phosphopeptide signal quantified as previously described. (19) Data plotted are from a combination of biological (n≥3) and technical (three spots) replicate analyses. One-sample t-tests were used to determine statistical significance.…”

Section: Methodsmentioning

confidence: 99%

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A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Placzek¹,

Plebanek²,

Lipchik³

et al. 2010

Analytical Biochemistry

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Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, since patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. While there are many ways to detect kinase activity, most lack the ability to ‘multiplex’ the analysis (to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live, intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Placzek¹,

Plebanek²,

Lipchik³

et al. 2010

Analytical Biochemistry

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“…Keck Foundation, Yale University. The ABI 4700 MALDI TOF/TOF was used to generate tandem mass spectrometer data using an ␣-cyano-4-hydroxycinnamic acid matrix as previously described (25). The samples used for direct infusion via static spray into the LTQ-FTICR were dissolved in 50% acetonitrile and 0.1% formic acid.…”

Section: Methodsmentioning

confidence: 99%

Sites of Intra- and Intermolecular Cross-linking of the N-terminal Extension of Troponin I in Human Cardiac Whole Troponin Complex

Warren

Kobayashi

Solaro

2009

Journal of Biological Chemistry

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706 -722) of the unique N-terminal region of human cardiac troponin I (hcTnI), predicted a possible intramolecular interaction near the basic inhibitory peptide. To explore this possibility, we generated single cysteine mutants (hcTnI-S5C and hcTnI-I19C), which were labeled with the hetero-bifunctional crosslinker benzophenone-4-maleimide. The labeled hcTnI was reconstituted to whole troponin and exposed to UV light to form cross-linked proteins. Reversed-phase high-performance liquid chromatography and SDS-PAGE indicated intraand intermolecular cross-linking with hcTnC and hcTnT. Moreover, using tandem mass spectrometry and Edman sequencing, specific intramolecular sites of interaction were determined at position Met-154 (I19C mutant) and Met-155 (S5C mutant) of hcTnI and intermolecular interactions at positions Met-47 and Met-80 of hcTnC in all conditions. Even though specific intermolecular cross-linked sites did not differ, the relative abundance of cross-linking was altered. We also measured the Ca 2؉ -dependent ATPase rate of reconstituted thin filament-myosin-S1 preparation regulated by either cross-linked or non-labeled troponin. Ca 2؉ regulation of the ATPase rate was lost when the Cys-5 hcTnI mutant was cross-linked in the absence of Ca 2؉ , but only partially inhibited with Cys-19 cross-linking in either the presence or absence of Ca 2؉ . This result indicates different functional effects of cross-linking to Met-154 and Met-155, which are located on different sides of the hcTnI switch peptide. Our data provide novel evidence identifying interactions of the hcTnI-N terminus with specific intra-and intermolecular sites. The human cardiac variant of troponin I (hcTnI)2 has structural and functional specializations that are related to its critical role in control of cardiac dynamics. These specializations include variations in amino acids that are significant factors in the response of the heart to: adrenergic stimulation (1), sarcomere length (2, 3), and pH (4, 5). An especially significant region of specialization is a unique N-terminal extension of 30 -32 amino acids, which contains serial serines at positions 23/24 that are substrates for kinases that control cardiac dynamics (6 -8). Despite its significance in control of cardiac function, molecular mechanisms of how the N-terminal human cardiac troponin I (N-hcTnI) region controls sarcomeric and cardiac function remain poorly understood. There is evidence that upon phosphorylation the interaction between the N-hcTnI and the N-lobe of N-hcTnC is weakened (9, 10). The structure of the N-hcTnI was missing in the crystal structure of cardiac troponin (11). However, we recently reported (12) the structure of the N-terminal peptide using NMR. Docking of this structure into the core troponin structure indicated the potential for a previously unappreciated intramolecular interactions of the N terminus with the regions at or near the highly basic inhibitory peptide region of cardiac troponin (12, 13). This interaction appeared plausible not only on the basis ...

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“…Towards developing a universally applicable approach to phosphorylation analysis they used these to determine subtleties in sequence-dependent differences for relative desorption/ionization efficiencies and to predict relative signal strengths for other peptide sequences [50].…”

Section: Phosphorylation Analysismentioning

confidence: 99%

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

Duncan¹,

Röder²,

Hunsucker³

2008

Briefings in Functional Genomics and Proteomics

191

162

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This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) timeof-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis. Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed.Keywords: quantification; quantitative analysis; MALDI; mass spectrometry; biomarkers OVERVIEWSince its inception in 1987 [1], matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) has been applied for the analysis of a wide range of biomolecules. Initial applications were almost exclusively for the qualitative analysis of biopolymers because MALDI-TOF MS provided a fast and accurate approach to molecular mass and purity information: Is my material the right mass and is it free from contaminants? Because of the speed of analysis, ease of use, relative low equipment cost, ease of data interpretation and limited potential for cross-contamination between samples/users, MALDI-TOF MS systems were considered walk-up instruments where investigators run their own samples and determine, within minutes, whether they were on the right track with their work.There were, however, two specific areas of application where MALDI-TOF MS was initially viewed as impractical: i.e. the analysis of low mass analytes and quantitative applications. Low mass analysis is complicated by the vast molar excess of the matrix that can swamp any analyte-specific signal in the low m/z region of the spectrum. Quantitative analysis was viewed as implausible because crystallization does not yield a uniform distribution of the analyte and matrix across the target surface and this gives rise to regions where the analyte signal is especially intense relative to other locations on the target surface. MALDI MS was therefore viewed as inherently irreproducible because, for a given amount of analyte loaded onto the target, the measured ion intensity varies.Subsequently, it has been shown that both these perceived limitations can be overcome and quantification can be routine, both for low and high mass analytes. Now, an extensive list of published applications includes quantification of amino acids, lipids, natural products, drugs, polymers, herbicides, metabolites, toxins, oligonucleotides, carbohydrates, peptides and proteins. (Selected applications from these areas are discussed at the end of this review.) Here, we focus on the quantitative applications of MALDI and illustrate applications relating to both low and high-molecular mass analytes. We also discuss the strategies for applying MALDI to relative and absolute quantification. While MALDI is most commonly combined with TOF analysers, other analyser options are possible and applications employing these are also presented. Our aim is not to provide an exhaustive catalogue of published applications, but to discuss the general principles and to illustrate the

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Investigating quantitation of phosphorylation using MALDI‐TOF mass spectrometry

Cited by 17 publications

References 15 publications

A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Sites of Intra- and Intermolecular Cross-linking of the N-terminal Extension of Troponin I in Human Cardiac Whole Troponin Complex

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

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