Protein Sample Preparation Protocol

All aqueous solutions were prepared with ultrapure water from a Milli-Q system (Millipore, Bedford, MA). All reagents were ACS grade from Sigma Aldrich (St. Louis, MO) unless otherwise specified. All protein and peptide solutions were stored at −20 °C unless otherwise specified. Hydrazine was prepared from the dichloride salt and the pH was adjusted with 10 M NaOH. Sodium acetate and glacial acetic acid were mixed to prepare a buffered 100 mM pH 4 solution. The pH of all protein and hydrazine solutions was determined using EMD Colorphast pH strips (Gibbstown, NJ) with the appropriate range of either 0-6 or 5-10, both with 0.5 pH unit accuracy. Hen egg white lysozyme (EC 3.2.1.14) was from Sigma Aldrich. Lysozyme concentrations were determined from the absorbance at 280 nm measured on a NanoDrop 1000 UV-Vis (Thermo Scientific, Wilmington, DE) and an estimated extinction coefficient of 37,970 M⁻¹cm⁻¹, based on the amino acid composition. Sequencing-grade-modified trypsin was from Promega (Madison, WI). Lissamine rhodamine B sulfonyl chloride was from Life Technologies (Grand Island, NY). Rhodamine aldehyde was synthesized from the commercial product as previously described.^{78 (link)}