Identification of the Oxidation States of the Active Site Cysteine in a Recombinant Protein Tyrosine Phosphatase by Electrospray Mass Spectrometry Using On-line Desalting Jon P. DeGnore 1 , Simone Ko ¨nig 1 , William C. Barrett 2 , P. Boon Chock 2 and Henry M. Fales 1 * 1 Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA 2 Laboratory of Biochemistry, Section of Metabolic Regulation, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA The oxidation state of the cysteine residue at the active site of human protein tyrosine phosphatase (PTP-1B) greatly affects its enzymatic activity. We wished to examine peroxide-treated preparations for modifications of this enzyme with electrospray mass spectrometry in order to determine the locations and oxidation states of the cysteines or other residues involved in the process. Since these reaction products contained large amounts of salts and buffers, they required desalting prior to analysis. Existing on- and off-line methods presented certain difficulties in handling and sample usage. Based on recent experience with direct syringe admission of sample, we developed a procedure as a simple, inexpensive alternative to full high- performance liquid chromatography systems that provides on-line desalting using only a few mL of sample. The method was applied to the analysis of oxidized PTP-1B preparations where conversion of cysteine 215 to both sulfinic and sulfonic acid residues was demonstrated. # 1998 John Wiley & Sons, Ltd. Received 29 June 1998; Revised 10 August 1998; Accepted 10 August 1998 Protein tyrosine phosphatases are a class of enzymes that catalyze the dephosphorylation of phosphotyrosine resi- dues. 1–6 The 37 kDa human protein tyrosine phosphatase (PTP-1B) member of this class of enzymes can become inactivated by various oxidation processes. It has been proposed 7 that, unlike many other proteins, PTP-1B’s activity does not require enzymatic post-translational modification but may be regulated by reversible oxida- tion/reduction involving the active site cysteine (at residue 215). Natural oxidation of these enzymes is conveniently modeled by use of hydrogen peroxide in a buffer system containing salts. 7,8 However, attempts to study such systems by electrospray mass spectrometry are hampered by the fact that these buffers severely suppress signal from the analyte, magnifying complications already present owing to the small amounts of modified enzymes formed. We therefore sought a procedure that would require minimum amounts of sample. Such methods include the Wilm and Mann nanospray technique 9,10 which uses disposable glass needles that are gold coated for electrical contact with the spray tip. These methods are reported to be quite effective with relatively high concentrations of salt and to provide analysis times of about 30 min to 1 h per mL. When required, a separate needle is used for off-line chromatographic desalting prior to transfer to another needle for electro- spraying. 11 Our experience with this system in this application is discussed below. Another method for off-line microscale sample desalting has been developed by Kussmann et al. 12 using GeLoader tips (Eppendorf, Hamburg, Germany) with POROS beads (PerSeptive Biosystems, Framingham, MA) where the exit end is constricted to retain the beads. In our hands the tips were found to be quite fragile and often not able to withstand the high pressure required to wash and elute the peptides. Off- line devices that can be connected to a syringe have also been fabricated with C-18 reverse phase packings for manual microcolumn chromatography. 13 Essentially, these are miniaturized guard columns used for standard liquid chromatography (LC) but generally they cannot handle the available small sample volumes. On-line desalting methods are also available and these have the immediate advantage of avoiding potentially wasteful sample transfers of mL volumes. Peptide trap cartridges containing C-18 support are often used in the sample loop position in the injection valve of ordinary LC systems with load volumes of 1 mL or above; obviously these do not meet our mL volume requirements. Other on- line desalting methods designed to handle low mL quantities of sample can be viewed as capillary liquid chromatography where the column dimensions are reduced to produce nL min 1 flow rates. However, the flow rates still are seldom less than 200 nL min 1 and this permits only 5 min of analysis time (elution time) for 1 mL of sample. 14 Compli- cated MS n experiments require substantially more time to determine fragmentation parameters, so a flow rate of 70 nL min 1 or less, providing approximately 14 min of analysis is more realistic. Another on-line method has been developed by Davis and Lee 15 which uses a tapered fused silica capillary needle (150 mm i.d. drawn to 5 mm i.d.) packed with C-18 column material. The column material was prevented from escaping the needle by use of a membrane frit inserted before the exit *Correspondence to: H. M. Fales, Laboratory of Biophysical Chemistry, Building 10, Room 7N 318, 9000 Rockville Pike, MD 20892, USA. CCC 0951–4198/98/201457–06 $17.50 # 1998 John Wiley & Sons, Ltd. RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 12, 1457–1462 (1998)