Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high-mass non-covalent complexes

Moniatte, Marc; Lesieur, Claire; Vécsey-Semjén, Beatrix; Buckley, J. Thomas; Pattus, Franc; Goot, F. Gisou van der; Dorsselaer, Alain Van

doi:10.1016/s0168-1176(97)00213-9

Cited by 47 publications

(52 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The peaks of the single subunits are the most intense peaks in this mass spectrum and the intensity of the (mostly nonspecific) hexamer fits the expected exponential decline of signal intensity with increasing mass. The outstanding intensity of the hexamer in the mass spectrum of Figure 1a, taken only from first shots onto a given spot, is interpreted as evidence of a specific noncovalent complex and representing the first-shot phenomenon [13][14][15]. Only subunits of APC could be observed using DHBs as matrix (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Localization of noncovalent complexes in MALDI-preparations by CLSM

Horneffer

Strupat

Hillenkamp

2006

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

The unambiguous detection of noncovalent complexes (NCCs) by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) is still a far cry from being routine. For protein NCCs such as their quaternary structure it has been reported that signals of the intact complex are only obtained for the first or at most the first few laser exposures of a given sample area. This observation was called the first-shot phenomenon. In the present study, this first-shot phenomenon has been investigated for the hexameric protein complex allophycocyanine (APC) by two independent methods, MALDI-MS with a (nearly) pH-neutral matrix 6-aza-2-thiothymine (6-ATT) and by imaging the fluorescence of the complex in APC-6-ATT preparations by confocal laser scan microscopy (CLSM). The intact APC heterohexamer loses its visible fluorescence upon dissociation into its subunits. Both methods consistently show that intact APC complexes are precipitated at the matrix crystal surface, but dissociate upon incorporation into the matrix crystals. (J Am Soc Mass Spectrom 2006, 17, 1599-1604

show abstract

Section: Resultsmentioning

confidence: 99%

Localization of noncovalent complexes in MALDI-preparations by CLSM

Horneffer

Strupat

Hillenkamp

2006

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

show abstract

“…Several major effects have been described to result in nonnative conditions for the noncovalent complexes: MALDI matrix [3][4][5], sample preparation [4,6,7], crystal morphology [4,8,9], pH of the solution [6, 10 -12], organic solvent [7,8], ionic strength [13,14], matrix/analyte ratio [15,16], speed of solvent evaporation [4], and sample concentration [9,15]. The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.Also, analyses by electrospray ionization (ESI) [21] are not carried out at physiological conditions as only solutions of very low ionic strength can be analyzed. However, ESI generates "colder" ions than MALDI and it keeps the sample in aqueous "biological" environment before ionization.…”

mentioning

confidence: 99%

“…The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.…”

mentioning

confidence: 99%

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

Yanes

Aviles

Roepstorff

et al. 2007

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

The difficulties to detect intact noncovalent complexes involving proteins and peptides by MALDI-TOF mass spectrometry have hindered a widespread use of this approach. Recently, "intensity fading MS" has been presented as an alternative strategy to detect noncovalent interactions in solution, in which a reduction in the relative signal intensity of low molecular mass binding partners (i.e., protease inhibitors) can be observed when their target protein (i.e., protease) is added to the sample. Here we have performed a systematic study to explore how various experimental conditions affect the intensity fading phenomenon, as well as a comparison with the strategy based on the direct detection of intact complexes by MALDI MS. For this purpose, the study is focused on two different protease-inhibitor complexes naturally occurring in solution, together with a heterogeneous mixture of nonbinding molecules derived from a biological extract, to examine the specificity of the approach, i.e., those of carboxypeptidase A (CPA) bound to potato carboxypeptidase inhibitor (PCI) and of trypsin bound to bovine pancreatic trypsin inhibitor (BPTI). Our results show that the intensity fading phenomenon occurs when the binding assay is carried out in the sub-M range and the interacting partners are present in complex mixtures of nonbinding compounds. Thus, at these experimental conditions, the specific inhibitor-protease interaction causes a selective reduction in the relative abundance of the inhibitor. Interestingly, we could not detect any gaseous noncovalent inhibitor-protease ions at these conditions, presumably due to the lower highmass sensitivity of MCP detectors. Several major effects have been described to result in nonnative conditions for the noncovalent complexes: MALDI matrix [3][4][5], sample preparation [4,6,7], crystal morphology [4,8,9], pH of the solution [6, 10 -12], organic solvent [7,8], ionic strength [13,14], matrix/analyte ratio [15,16], speed of solvent evaporation [4], and sample concentration [9,15]. The effect of some instrumental parameters in the detection of complexes has also been studied; extraction delay time, positive/negative ion mode, linear/reflector and acceleration mode were found to be of minor importance [7,17], whereas laser pulse energy [8,17,18] and the number of laser shots (i.e., "first shot phenomenon") [4,8,13,19,20] was reported to be a decisive factor in a number of cases.Also, analyses by electrospray ionization (ESI) [21] are not carried out at physiological conditions as only solutions of very low ionic strength can be analyzed. However, ESI generates "colder" ions than MALDI and it keeps the sample in aqueous "biological" environment before ionization. For these reasons, ESI has been used in numerous studies to detect noncovalent complexes [22].Relatively few cases have been reported where specific intact noncovalent complexes were successfully observed with MALDI. Besides the frequent dissociation of noncovalent complexes due to the experimental conditions employed, a further...

show abstract

“…This work showed the feasibility of directly measuring hydrogen/deuterium exchange kinetics of detergent-solubilized proteins in solution by ES mass spectrometry and thereby obtaining structural information. Although ES has now been extensively used in the direct study of noncovalent interactions, the application of matrix-assisted laser desorption ionization (MALDI) in this area of research been more restricted, although it should be noted that Van Dorsselaer et al (33,34) have reported MALDI data on the subunit stoichiometry of high mass noncovalent complexes of membrane proteins.…”

mentioning

confidence: 99%

Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

Lengqvist

Svensson

Evergren

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer⅐glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys 49 (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144 -15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.Membrane proteins are estimated to constitute about onethird of the total proteome (1) and have important and diverse roles in biology, including functions as cell-surface receptors, ion channels, and transporters and in cell adhesion. However, because of the difficulty in producing crystals of detergentsolubilized proteins for x-ray crystallography, only ϳ1% of the unique protein structures so far solved at high resolution are of membrane proteins (2). Integral membrane proteins thus pose an important challenge in structural biology.Microsomal glutathione transferase-1 (MGST1) 1 is a member of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily. At present, other mammalian members of the MAPEG family are two glutathione transferases/peroxidases (MGST2 and MGST3), 5-lipoxygenase-activating protein, leukotriene C 4 synthase, and microsomal prostaglandin E 2 synthase (the closest relative to MGST1) (3). MGST1 functions both as a glutathione transferase and as a peroxidase, thus potentially protecting cells from reactive compounds and oxidative stress (3, 4). The quaternary structure of MGST1 has previously been investigated by electron crystallography (5, 6), radiation inactivation (7), cross-linking (8), and hydrodynamic (9) studies and has been suggested to be a homotrimer both in the microsomal membrane and in the purified form. Electrospray (ES) mass spectrometry has shown that the enzyme is partly N-acetylated (10). Interestingly, equilibrium dialysis (11) and stopped-flow active-site titrations (12) have indicated that the MGST1 homotrimer binds only one molecule of ...

show abstract

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high-mass non-covalent complexes

Cited by 47 publications

References 38 publications

Localization of noncovalent complexes in MALDI-preparations by CLSM

Localization of noncovalent complexes in MALDI-preparations by CLSM

Exploring the “intensity fading” phenomenon in the study of noncovalent interactions by MALDI-TOF mass spectrometry

Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

Contact Info

Product

Resources

About