SearchXLinks. A Program for the Identification of Disulfide Bonds in Proteins from Mass Spectra

Wefing, S.; Schnaible, Volker; Hoffmann, Daniel

doi:10.1021/ac051634x

Cited by 39 publications

(37 citation statements)

References 39 publications

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“…Mass spectra were obtained either in linear or positive reflector mode using a Voyager 4700 MALDI/TOF mass spectrometer (Applied Biosystems). The mass spectrometry data were analyzed by Data Explorer (Applied Biosystems), FindPept (ExPASy proteomics server), and SearchXlinks (20).…”

Section: Ide Protein Expression and Purification-mentioning

confidence: 99%

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Manolopoulou

Guo

Malito

et al. 2009

Journal of Biological Chemistry

174

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Insulin is a hormone vital for glucose homeostasis, and insulindegrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our timedependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N-and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (ϳ100 nM) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages. IDE3 is an ϳ110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (ϳ100 nM) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-␤ (A␤), a peptide vital to the progression of Alzheimer disease (7,8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of A␤ (5, 9 -12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter-and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the d...

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Section: Ide Protein Expression and Purification-mentioning

confidence: 99%

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Manolopoulou

Guo

Malito

et al. 2009

Journal of Biological Chemistry

174

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“…Indeed, a disulfide bond tends to be reduced during the ISD process [46] and therefore acts as a quencher of the fragmentation of neighboring amino acids. This observation was exploited by Schnaible et al to localize disulfide bonds on HPLC-separated peptides [47,48]. Moreover, PTMs that do not interfere with the ISD fragmentation of the peptide backbone can be studied by ISD as well.…”

Section: Maldi-isd and Ptmsmentioning

confidence: 99%

MALDI In-Source Decay, from Sequencing to Imaging

Debois

Smargiasso

Demeure

et al. 2012

Topics in Current Chemistry

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Matrix-assisted laser desorption/ionization (MALDI) is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans, etc). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa, thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS3 can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications (PTMs) studies, and finally review MALDI-ISD tissue imaging applications.

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“…The total peptide masses were compared for (1) each individual protein with no BS 3 treatment (negative control), (2) each individual protein plus BS 3 (showing intramolecular cross-links), and (3) the two proteins mixed together plus BS 3 (to deduce intermolecular cross-links). The resulting masses were analyzed through the online software Automated Spectrum Assignment Program (ASAP) (Lee et al 2007) and SearchXlinks (Wefing et al 2006). Only masses that uniquely identified (Boon et al 2006).…”

Section: Prp8pmentioning

confidence: 99%

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

Grainger¹,

Barrass²,

Jacquier³

et al. 2009

RNA

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In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.

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SearchXLinks. A Program for the Identification of Disulfide Bonds in Proteins from Mass Spectra

Cited by 39 publications

References 39 publications

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

MALDI In-Source Decay, from Sequencing to Imaging

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

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