5 research outputs found
Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers
Quantitative
measurement of chemically cross-linked proteins is
crucial to reveal dynamic information about protein structures and
protein–protein interactions and how these are differentially
regulated during stress, aging, drug treatment, and most perturbations.
Previously, we demonstrated how quantitative in vivo cross-linking
(CL) with stable isotope labeling of amino acids in cell culture (SILAC)
enables both heritable and dynamic changes in cells to be visualized.
In this work, we demonstrate the technical feasibility of proteome-scale
quantitative in vivo CL–MS using isotope-labeled protein interaction
reporter (PIR) cross-linkers and <i>E. coli</i> as a model
system. This isotope-labeled cross-linkers approach, combined with
Real-time Analysis of Cross-linked peptide Technology (ReACT) previously
developed in our lab, enables the quantification of 941 nonredundant
cross-linked peptide pairs from a total of 1213 fully identified peptide
pairs in two biological replicate samples through comparison of MS<sup>1</sup> peak intensity of the light and heavy cross-linked peptide
pairs. For targeted relative quantification of cross-linked peptide
pairs, we further developed a PRM-based assay to accurately probe
specific site interaction changes in a complex background. The methodology
described in this work provides reliable tools for both large-scale
and targeted quantitative CL–MS that is useful for any sample
where SILAC labeling may not be practical
Conditional Fragment Ion Probabilities Improve Database Searching for Nonmonoisotopic Precursors
Stochastic, intensity-based precursor
isolation can result in isotopically
enriched fragment ions. This problem is exacerbated for large peptides
and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous
labeling strategies result in the isolation of peptide ions composed
of many distinct structural isomers. Unfortunately, existing proteomics
search algorithms do not account for this variability in isotopic
incorporation, and thus often yield poor peptide and protein identification
rates. We sought to resolve this shortcoming by deriving the expected
isotopic distributions of each fragment ion and incorporating them
into the theoretical mass spectra used for peptide-spectrum-matching.
We adapted the Comet search platform to integrate a modified spectral
prediction algorithm we term Conditional fragment Ion Distribution
Search (CIDS). Comet-CIDS uses a traditional database searching strategy,
but for each candidate peptide we compute the isotopic distribution
of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS
identified more confident peptide spectral matches and higher protein
sequence coverage compared to traditional theoretical spectra generation,
with the magnitude of improvement largely determined by the amount
of labeling in the sample
Quantification of BSA cross-linked peptide pairs with Skyline.
<p><b>A.</b> MS2 spectrum for the cross-linked peptide pair linking residues K235-K28 (ALK<sup>235</sup>AWSVAR_DTHK<sup>28</sup>SEIAHR), obtained from a 500 ng injection of cross-linked BSA digest. <b>B.</b> Extracted ion chromatograms for the PRM transitions observed for the cross-linked peptide pair in A. <b>C.</b> Skyline generated bar plot illustrating the normalized peak areas for the cross-linked peptide pair linking K28-K235. Peak areas are shown for triplicate analyses of varying injection amounts (100, 200, 500, and 1000 ng cross-linked BSA digest). Bars are color coded to indicate the contribution of each individual transition to the total peak area and match the color scheme in panel B. </p
Cross-laboratory quantification of BSA-BDP cross-linked peptide pairs.
<p><b>A.</b> Average cross-linked peptide response curve comparing data collected in the Bruce Lab and CSHL for 30 cross-linked peptide pairs. <b>B.</b> Scatter plot illustrating the R<sup>2</sup> values for linear regression analysis of the data shown in A and B.</p
Experimental outline.
<p><b>A.</b> Biological samples are prepared for qXL-MS comparing two or more conditions. The samples are treated with chemical cross-linker either as (1) a mixed sample if SILAC labeling was used or (2) as separate samples if carrying out a label free experiment or using isotopically labeled cross-linkers. Following the cross-linking reaction proteins are extracted, enzymatically digested, and subjected to various strategies (i.e. strong cation exchange and affinity chromatography) for enrichment cross-linked peptide pairs. <b>B.</b> LC-MS analysis of samples enriched for cross-linked peptide pairs is carried out. This consists of reversed phase chromatographic separation by LC followed by analysis by MS. The mass spectrometer is operated in PRM mode where an inclusion list of <i>m/z</i> values for the precursor ions of interest is used to target specific cross-linked peptides. The PRM mass spectrometric analysis used here consists of three steps including isolation of precursor ions, fragmentation by collision with neutral gasses, and detection of mass to charge ratios of the resulting fragment ions. <b>C)</b> Resulting MS2 data are converted into transition lists and imported into Skyline for analysis.</p