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Publisher Summary This chapter discusses results of a study focusing on characterization of the proteins c-kit ligand and dihydrofolate reductase (DHFR) by electrospray mass spectrometry (ESI). Human KL expressed in E.coli was purified in 8 M urea/40 mM Tris–HCl, pH 7 on DEAE-Sepharose Fast Flow. After 100-fold dilution and dialysis against PBS followed by 40 mM Tris–HCI, the protein was passed over Q-Sepharose Fast Flow (Pharmacia). The fractions of the main peak eluting with about 150 mM NaCl were pooled and applied to a Superdex 200 (Pharmacia) column. KL eluted with an apparent molecular weight of about 35 to 40 kD. The protein was desalted on an Aquapore C4 column for ESI–MS use. Peptides with short elution times were determined by flow injection of the complete digest into the ESI–MS. The results indicated that the combination of LC with ESI–MS was very useful for the interpretation of tryptic digests and for the elucidation of disulfide bridges.