Isotope Signatures Allow Identification of Chemically Cross-Linked Peptides by Mass Spectrometry: A Novel Method to Determine Interresidue Distances in Protein Structures through Cross-Linking

Knowledge of protein structures and protein−protein interactions is essential for understanding of biological processes. Recent advances in protein cross-linking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we report a novel method to rapidly and accurately identify cross-linked peptides based on their unique isotope signature when digested in the presence of H218O. This method overcomes the need for specially synthesized cross-linkers and/or multiple MS runs required by other techniques. We validated our method by performing a “blind” analysis of 5 proteins/complexes of known structure. Side chain repacking calculations using Rosetta show that 17 of our 20 positively identified cross-links fit the published atomic structures. The remaining 3 cross-links are likely due to protein aggregation. The accuracy and rapid throughput of our workflow will advance the use of protein cross-linking in structural biology

Kojak: Efficient Analysis of Chemically Cross-Linked Protein Complexes

Protein chemical cross-linking and mass spectrometry enable the analysis of protein–protein interactions and protein topologies; however, complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein–protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with or without heavy isotope labels. Kojak was used to analyze both novel and existing data sets and was compared to existing cross-linking algorithms. The algorithm provided increased cross-link identifications over existing algorithms and, equally importantly, the results in a fraction of computational time. The Kojak algorithm is open-source, cross-platform, and freely available. This software provides both existing and new cross-linking researchers alike an effective way to derive additional cross-link identifications from new or existing data sets. For new users, it provides a simple analytical resource resulting in more cross-link identifications than other methods

Discovery and Visualization of Uncharacterized Drug–Protein Adducts Using Mass Spectrometry

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools

Discovery and Visualization of Uncharacterized Drug–Protein Adducts Using Mass Spectrometry

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools

Dimerization of Stu2 is required for kinetochore association.

A) Schematic of Stu2’s domain architecture and corresponding deletions. Note: Internal deletions (i.e. not at the N- or C-terminus) were made by inserting a linker, denoted by a dashed line. B) Exponentially growing stu2-AID cultures expressing an ectopic copy of STU2 (STU2WT, SBY13901; stu2TOG1Δ, SBY13904; stu2TOG2Δ, SBY13907; stu2SK-richΔ, SBY13913; stu2ccΔ, SBY13916; or stu2C-termΔ(855–888), SBY14269) that also contained Dsn1-6His-3Flag were treated with auxin 30 min prior to harvesting. Protein lysates were subsequently prepared and kinetochore particles were purified by α-Flag immunoprecipitation (IP) and analyzed by immunoblotting. C) Cross-linking mass spectrometry analysis reveals interactions between recombinant Stu2-eGFP and the Ndc80 complex. Cross-links formed with EDC between Stu2 and the Ndc80 complex are depicted by red lines. Bar diagrams with structural features of Ndc80 and Stu2-eGFP proteins are included. (CH: calponin homology; HP: hairpin; TOG: tumor overexpressed gene; SK-rich: regions with stretches of sequences rich in Serine, Glycine, Lysine). For clarity, cross-links within Ndc80 complex proteins and those involving Stu2 fusion regions (extreme N-terminus and eGFP) are shown in grey. Data are shown for peptides with Percolator assigned q-values ≤ 0.05 and a minimum of 2 PSMs. D) Exponentially growing Stu2-AID cultures with an ectopic copy of STU2 (STU2WT, SBY13901; stu2ccΔ, SBY13916; or stu2ccΔ::bZIP, SBY13935) or without an ectopic allele (no covering allele, SBY13772) that also contained Dsn1-6His-3Flag were treated with auxin 30 min prior to harvesting. Protein lysates were subsequently prepared and kinetochore particles were purified by α-Flag immunoprecipitation (IP) and analyzed by immunoblotting. E) Wild-type (SBY3), stu2-AID (no covering allele, SBY13772) and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus (STU2WT, SBY13901; stu2ccΔ, SBY13916; stu2ccΔ::bZIP, SBY13935) were serially diluted (5-fold) and spotted on plates containing either DMSO or 500 μM auxin. Refer to S2 Fig for a similar analysis of all alleles examined.</p

Kinetochore-binding by Stu2 is required for enhancing Ndc80c-microtubule attachment stability.

A) Schematic of optical trap assay. Dynamic microtubules are grown from coverslip-anchored seeds. Beads linked to Ndc80c via Spc24 are manipulated using an optical trap to exert applied force across the Ndc80c-microtubule interface in the presence or absence of purified Stu2. B) Mean rupture forces for Ndc80c-linked beads untreated or incubated with free Stu2WT or Stu2ccΔ. Error bars represent SEM (n = 22–31 events). P value was determined using a two-tailed unpaired t test. All measurements were conducted within the same experimental set, but the data for Ndc80c alone and in the presence of Stu2WT are reported previously in [10]. C) Attachment survival probability versus force for the data in (B).</p

Discovery and Visualization of Uncharacterized Drug–Protein Adducts Using Mass Spectrometry

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life-threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics approaches incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with three drug/protein combinations and apply our label-free workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing label-free tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools

Mutants in Stu2 and Aurora B show a synergistic biorientation defect.

A) Wild-type (SBY3), stu2-AID (no covering allele, SBY13772) and stu2-AID cells expressing various STU2-3V5 alleles from an ectopic locus (STU2WT, SBY13903; stu2ccΔ, SBY13918) or also containing an ipl1-321 allele (stu2ccΔ ipl1-321, SBY17100) or ipl1-321 alone (SBY630) were serially diluted (5-fold) and spotted on YPD or 5 μg/ml benomyl plates containing either DMSO or 500 μM auxin and incubated at 23°C (permissive) or 30°C (semi-permissive). B) Exponentially growing stu2-AID mad3Δ cells containing or lacking an ipl1-321 allele and also an ectopically expressed STU2 allele (STU2WT IPL1, SBY18242; STU2WT ipl1-321, SBY18244; stu2ccΔ IPL1, SBY18246; stu2ccΔ ipl1-321, SBY18248) that also contained a fluorescently labeled CEN3 were arrested in G1 at the permissive temperature (23°C) and subsequently released from the G1 arrest into auxin containing media at a semi-permissive temperature (30°C). Quantification of chromosome mis-segregation in anaphase is shown. Error bars represent SD of three independent experiments; n = 100 cells for each experiment. p value was determined using a two-tailed paired t test. C) Model: Kinetochore-associated Stu2 confers tension sensitivity to kinetochore-microtubule attachments and is required for the establishment of bioriented attachments in vivo. Stu2 and Aurora B function together to release (destabilize) low tension bearing/incorrect kinetochore-microtubule attachments in cells. At high tension, a second function of Stu2 serves to stabilize correct attachments.</p

Kinetochore-binding deficient Stu2 exhibits a biorientation defect and spindle checkpoint-dependent cell cycle delay.

A) Exponentially growing stu2-AID cdc20-AID cultures with an ectopically expressed STU2 allele (STU2WT, SBY17369; stu2ccΔ, SBY17371) or without an ectopic allele (no covering allele, SBY17367) that also contained MTW1-3GFP (kinetochore) and SPC42-CFP (spindle pole; marked with white arrows) were treated with auxin for 2 h to arrest cells in metaphase. Representative images for each are shown. White bars represent 2 μm. B) Quantification of Mtw1 localization from (A). Error bars represent SD of two independent experiments; n = 100 cells for each experiment. C) Kinetochore distribution (distance between bi-lobed kinetochore clusters) and spindle length (spindle pole-to-pole distance; Fig 2B) was measured for cells described in (A). n = 80–105 cells; p values were determined using a two-tailed unpaired t test. D) stu2-AID cells with an ectopically expressed STU2 allele with and without a spindle checkpoint mutation (STU2WT MAD3, SBY17527; stu2ccΔ MAD3, SBY17560; STU2WT mad3Δ, SBY17668; stu2ccΔ mad3Δ, SBY17669) that also contained a fluorescently labeled CEN8 were released from a G1 arrest into auxin containing media. Cell cycle progression determined by the accumulation of binucleate cells. Shown is a representative experiment. E) Quantification of chromosome mis-segregation in anaphase (percent of binucleate cells with a fluorescently labeled CEN8 signal in only one of the two nuclei) from (D). Error bars represent SD of two independent experiments; n = 200 cells for each experiment.</p

Kinetochore-associated Stu2 is not required for maintenance of biorientation.

A) Exponentially growing stu2-AID cdc20-AID cultures with an ectopically expressed STU2 allele (STU2WT, SBY17748; stu2ccΔ, SBY17750) or without an ectopic allele (no covering allele, SBY17708) that also contained a fluorescently labeled CEN8 were treated with auxin for 2 h to arrest cells in metaphase. Percentage of cells that display two distinct GFP foci (i.e. bioriented CEN8) was quantified. Shown is the average of two independent experiments, error bars represent SD (n ≧ 100 cells). B) Schematic of experiment in (C). C) Exponentially growing stu2-AID pMET-CDC20 cells that contained a fluorescently labeled CEN3 and an ectopically expressed STU2 allele (STU2WT, SBY18370; stu2ccΔ, SBY18371) or without an ectopic allele (no covering allele, SBY18359) were arrested in metaphase by the addition of methionine for 3 h. Once arrested in metaphase, auxin was added to degrade the Stu2-AID protein and the percentage of cells that display two distinct GFP foci (i.e. bioriented CEN3) was quantified over time. Error bars represent SD of three independent experiments; n = 100 cells for each time point.</p