Sample Multiplexing with Cysteine-Selective Approaches: Cysdml and Cpilot

Supplemental Information for:

Sample Multiplexing with Cysteine-selective Approaches: cysDML and cPILOT

LiqingGu, Adam R. Evans and Renã A. S. Robinson*

Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260 USA

Supplemental Figure S1. Optimization of on-resin dimethylation.

Supplemental Figure S2. On-resin loading capacity of cysDML experiment.

Supplemental Figure S3. Base peak chromatograms for an example cysDML experiment (one biological replicate).

Supplemental Figure S4. Distribution of m/z spaces between light and heavy species of all PSMs in cPILOT experiment and charge states.

Supplemental Table S1. List of proteins identified in cysDML experiment.

Supplemental Table S2. List of peptides identified in cysDML experiment.

Supplemental Table S3. List of proteins identified in cPILOT experiment.

Supplemental Table S4. List of peptides identified in cPLOT experiment.

Supplemental Figure Captions

Supplemental Figure S1. a) Plot of on-resin dimethylation labeling efficiency (calculated based on spectral counts) of N-terminal and Lys residues usingvarious reaction times; b) labeling efficiency of N-terminal and Lys residues using 25mM NaBH3CN and 60mM NaBH3CN with a one-hour incubation time.

Supplemental Figure S2. a) Scatter plot of protein ratios measured in cysDML experiment designed to have theoretical ratios of 1:1 for light and heavy labeled peaks. Horizontal lines represent average±2×standard deviation (µ±2σ) and the numbers of quantified proteins within each region are labeled. b) Box plot of measured ratios in cysDML dynamic range experiment. The amount of peptide labeled with the light tag is fixed at 100µg, while the amount of peptide labeled with the heavy tag varies as follows: 12.5µg, 25µg, 50µg, 100µg, 200µg and 400µg. The theoretical ratios of light and heavy labeled peaks are designed to 0.125, 0.25, 0.5, 1, 2 and 4.

Supplemental Figure S3. Base peak chromatograms for an example cysDML experiment (one biological replicate). Samples are injected a total of seven times. The first injection is analyzed with a full m/z range of 350-1700. The subsequent injections use gas phase fractionation (GPF) such that data are acquired over the m/z ranges of 350-800, 785-975 and 960-1700, and repeated twice. The top 15 most intense ions are selected and fragmented in each run.

Supplemental Figure S4. Pie chart of PSM numbers with different a) m/z spaces between light and heavy species in cPILOT experiment and b) charge states.