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 (ALK²³⁵AWSVAR_DTHK²⁸SEIAHR), 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 R² 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