Product ion scanning using a Q‐q‐Q<sub>linear ion trap</sub> (Q TRAP<sup>TM</sup>) mass spectrometer

Hager, James W.; Blanc, J. C. Yves Le

doi:10.1002/rcm.1020

Cited by 138 publications

(102 citation statements)

References 19 publications

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“…Alternatively, precursor ion scanning for m/z 79 was performed online during LC-ESMS using a Sciex API 4000 triple quadrupole-linear ion trap (29). Tryptic peptides from IQGAP1 were separated by nanobore (75 m inner diameter) reversed phase HPLC using acetonitrile/ water gradients flowing at 0.3 l/min.…”

Section: Methodsmentioning

confidence: 99%

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

McNulty

Marler

et al. 2005

Journal of Biological Chemistry

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In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, ␤-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase C⑀, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.Initially identified 10 years ago, IQGAP1 has been shown to participate in several fundamental cellular processes (for reviews see Refs 1 and 2). These include cell-cell attachment, ␤-catenin-mediated transcription, cell migration, regulation of actin, microtubule function, the mitogen-activated protein kinase cascade, and Ca 2ϩ /calmodulin signaling (2-4). IQGAP1 is a component of these diverse functions via direct interactions with multiple target proteins that are mediated by a number of protein interaction motifs in IQGAP1. These include the following: a calponin homology domain, responsible for actin binding (5, 6); a WW motif, which is necessary for the association of extracellular signal-regulated kinase 2 (a component of the mitogen-activated protein kinase pathway) (4); a calmodulinbinding IQ domain (5, 7); and a RasGAP-related domain that binds the small GTPases Cdc42 and Rac1 (5, 8). In addition, IQGAP1 binds to and regulates the functions of E-cadherin, ␤-catenin, and CLIP-170 (9 -12).Accumulating evidence reveals an important role for IQGAP1 in cytoskeletal function. The cytoskeleton of eukaryotic cells comprises several elements, including actin, microtubules, and intermediate filaments. Numerous proteins interact with the actin cytoskeleton...

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Section: Methodsmentioning

confidence: 99%

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

McNulty

Marler

et al. 2005

Journal of Biological Chemistry

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“…Neutral loss collision energy profiling for the method development of the multiple neutral loss monitoring (MNM) scan was performed on a 2000 Q-TRAP instrument (Applied Biosystems/MDS Sciex, Framingham, MA) in nanospray mode using the enhanced product ion (EPI) scan, which essentially traps all product ions utilizing Q3 as a linear ion trap [6]. Neutral loss collision energy profiles of all neutral losses were collected by varying the collision energy from 5 to 150 eV in increments of 2 V for the collision energy acceleration potential.…”

Section: Mass Spectrometrymentioning

confidence: 99%

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Hoffman

Sniatynski

Rogalski

et al. 2006

J. Am. Soc. Mass Spectrom.

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Post-translational modifications of proteins are involved in determining the activity of proteins and are essential for proper protein function. Current mass spectrometric strategies require one to specify a particular type of modification, in some cases also a particular charge state of a protein or peptide that is to be studied before the actual analysis. Due to these requirements, most of the modifications on proteins are not considered in such an experiment and, thus, a series of similar analyses need to be performed to ensure a more extensive characterization. A novel scan strategy has been developed, multiple neutral loss monitoring (MNM), allowing for the comprehensive screening of post-translational modifications (PTM) on proteins that fragment as neutral losses in a mass spectrometer. MNM method parameters were determined by performing product ion scans on a number of modified peptides over a range of collision energies, providing neutral loss energy profiles and optimal collision energies (OCE) for each modification, supplying valuable information pertaining to the fragmentation of these modifications and the necessary parameters that would be required to obtain the best analysis. As the optimal collision energy was highly dependent on the type of modification and the charge state of the peptide, the MNM scan was operated with a collision energy gradient. Autocorrelation analyses identified the type of modification, and convolution mapping analyses identified the associated peptide. The MNM scan with the new collision energy parameters was successfully applied to a mixture of four modified peptides in a BSA digest. The implementation of this technique will allow for comprehensive screening of all modifications that fragment as neutral losses. (J Am Soc Mass Spectrom 2006, 17, 307-317)

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“…In fact, it must be emphasized that, for the fullscan spectra acquisition mode, the IT is definitely more sensitive than a linear mass filter, but the opposite is true for MRM mode also if the new linear ion trap seems to reach similar performances (Hager & Yves Le Blanc, 2003).…”

Section: Sensitivitymentioning

confidence: 99%

“…It is also possible to use the MRM as well as precursor-and neutral-loss scanning with the enhanced capability of the linear ion trap (Hager & Yves Le Blanc, 2003).…”

Section: Linear Quadrupole Ion Trapmentioning

confidence: 99%

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

Cristoni

Bernardi

2003

Mass Spectrometry Reviews

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I. Introduction 370 II. Techniques That Are Usually Employed in the Study of Bioorganic Macromolecules 371 A. Proteins and Peptides 371 B. Oligonucleotides 374 C. Oligosaccharides and Glycoconjugates 375 D. Various Complexes (DNA–Protein, Antigen–Antibody, Macromolecules–Metals) 377 III. Common Problems and Developments in the Analysis of Bioorganic Macromolecules by Mass Spectrometry 377 A. Ionization Source Problems and Developments 377 1. Sensitivity 377 2. Problems with Buffers 379 3. Multicharge Effect 381 B. Mass Analyzer Problems and Developments 381 1. Linear Dynamic Range 381 2. Tandem Mass Spectrometry 382 3. Sensitivity 382 4. Resolution 383 5. Mass Accuracy 383 IV. New Promising Technologies 384 A. Interfaces and Ionization Sources 384 1. Improvements in the Coupling of Liquid‐Phase Separation Systems to Mass Spectrometry 384 2. AP‐MALDI 384 3. Deprotonant Agents 385 4. Sonic Spray Ionization 388 5. APCI without Corona Discharge 391 B. Mass Analyzers 393 1. Linear Quadrupole Ion Trap 394 2. TOF Analyzers with New Detectors 395 3. Multiple Mass Analyzers 396 V. Software for Data Treatment 397 VI. Conclusions and Future Developments 399 Acknowledgments 400 References 400 In recent years, mass spectrometry has been increasingly used for the analysis of various macromolecules of biological, biomedical, and biochemical interest. This increase has been made possible by two key developments: the advent of electrospray ionization (ESI) and matrix‐assisted laser desorption ionization (MALDI) sources. The two new techniques produce a significant increase in mass range and in sensitivity that led to the development of new applications and of new analyzer designs, software, and robotics. This review, apart from the description of the status of mass spectrometry in the analysis of bioorganic macromolecules, is mainly devoted to the illustration of the more recent promising techniques and on their possible future evolution. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:369–406, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10062

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Product ion scanning using a Q‐q‐Q_{linear ion trap} (Q TRAP^TM) mass spectrometer

Cited by 138 publications

References 19 publications

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

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Product ion scanning using a Q‐q‐Qlinear ion trap (Q TRAPTM) mass spectrometer

Cited by 138 publications

References 19 publications

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

Multiple neutral loss monitoring (MNM): A multiplexed method for post-translational modification screening

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

Contact Info

Product

Resources

About

Product ion scanning using a Q‐q‐Q_{linear ion trap} (Q TRAP^TM) mass spectrometer