Measurement of Protein Sulfenic Acid Content

Materials

• Putative sulfenic acid–containing protein, purified and in a neutral pH buffer (pH 6.5 to 7.5)

• Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 (see APPENDIX 2A) or equivalent/1 mM EDTA, or other pH 7 buffer

• 100 mM NBD chloride (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) in DMSO (see recipe)

• Acetonitrile

• Formic acid

• Anaerobic quartz cuvette (if required) and regular quartz cuvette

• Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)

• UV-visible scanning or diode-array spectrophotometer

• Electrospray ionization mass spectrometer (ESI-MS) or access to a fee-for-service facility

• Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)

• Additional reagents and equipment for preparing proteins containing sulfenic acid (Support Protocol 1) and determining protein concentration by a non-NBD-affected assay (e.g., Detergent-Compatible Protein Assay Kit, Bio-Rad)

Prepare protein

• 1

  Prepare at least 1 ml of a 15 μM (monomer or target thiol group concentration) solution of the putative sulfenic acid–containing protein in neutral pH buffer, using the method described in Support Protocol 1 or by other methods established by functional analyses.

  For example, in a typical reaction, add 3 μl of 8 mM hydrogen peroxide to 0.5 ml of 45 μM protein (Support Protocol 1).

  Oxidized proteins should be prepared immediately before use.

Modify protein with NBD chloride

• 2

  To the protein of interest (with or without oxidant or reductant pretreatment, as experimental and/or controls samples) add a small volume of 100 mM NBD chloride, either directly or through the sidearm of the anaerobic cuvette. Incubate 10 to 30 min at room temperature.

  More reagent gives a faster reaction, allowing pseudo-first-order kinetics to be followed if any spectral changes are observed; therefore, for most experiments, an excess of at least 10-fold NBD over protein thiol and sulfenic-acid groups is desirable.

  Anaerobic conditions may be required to stabilize the sulfenic acid during its generation, as described in Support Protocol 1; if so, this step should also be conducted anaerobically.

  Denaturation of the protein may be required in this step if the sulfenic acid is not accessible (see the Troubleshooting section in the Commentary).

  Thus, continuing with the example from the step 1 annotation, add 3 μl NBD chloride solution and incubate 10 to 30 min at room temperature. After modification, the protein in the cuvette can be exposed to air for the ultrafiltration step that follows.

Analyze modified protein by spectroscopy

• 3

  Transfer the protein-NBD solution to an ultrafiltration unit of appropriate molecular weight cutoff (MWCO). Remove the unreacted NBD chloride as follows:

  1. Add 6 ml neutral pH buffer.

  2. Concentrate to 50 μl according to manufacturer’s instructions.

  3. Repeat two to three times.

     Attention must be paid to the stated MWCO of the device so that the protein of interest will be retained. With each concentration cycle, the flow-through solution (filtrate) is removed, and fresh neutral pH buffer is added to dilute the protein again, for a total of two to three concentration/redilution cycles.

     Either Centricon ultrafiltration units, which allow 2 ml to be concentrated to 40 to 50 μl, or larger units offered by several other suppliers (e.g., Orbital Biosciences or Vivascience), which allow 6 to 7 ml to be concentrated to ~10 to 50 μl, can be used. In the case of the latter, the ability to concentrate more solution at once and to use swinging bucket rotors, rather than fixed-angle rotors, can speed up the washing process.

     Alternatively, the modified protein can be isolated (without ultrafiltration) by HPLC in tandem with injection into a mass spectrometer.

• 4

  Check for the presence of free NBD in the filtrate by determining the absorbance at 343 nm. Repeat step 3 until no free reagent is detected (i.e., A₃₄₃ is <0.02).

• 5

  Optional: Assess the protein concentration using a protein assay that is not affected by the presence of the NBD—e.g., the Detergent-Compatible (DC) Protein Assay Kit from Bio-Rad.

  The A₂₈₀ (APPENDIX 3G) now has a contribution from the presence of NBD and therefore cannot be used to determine the protein concentration. Colorimetric assays monitoring wavelengths >500 nm may work (see APPENDIX 3I).

• 6

  Remove the concentrated protein from the ultrafiltration unit and bring to approximately the original concentration with neutral pH buffer, assuming there has been no loss of protein during the ultrafiltration step.

  Protein recovery from the ultrafiltration devices can be tested independently with unmodified protein.

• 7

  Perform a spectral scan from ~250 to 700 nm in a quartz cuvette to assess of the nature of the modification.

  Thiol adducts with NBD give a peak at 420 nm with an extinction coefficient of 13,000 M⁻¹ cm⁻¹ (Birkett et al., 1970). Sulfenic acid adducts with NBD give a peak at 347 nm with a similar extinction coefficient (Ellis and Poole, 1997a).

Prepare modified protein for ESI-MS

• 8

  Wash the modified protein sample at least three times by complete concentration and redilution with water (as described in step 3) to remove excess reagent and buffer components.

• 9

  Prepare 1 nmol modified protein in 100 μl water. Add an equal volume of acetonitrile and 2 μl formic acid before injection. Analyze by ESI-MS.

  This amount of sample is far above that required for the analysis by most modern ESI-MS instruments. Optimization of sample analyses may involve changing the acetonitrile concentration and/or using a volatile buffer such as ammonium bicarbonate instead of water during the washing step (Fuangthong and Helmann, 2002).

  Alternatively, the modified protein can be isolated (without ultrafiltration) by HPLC in tandem with injection into the mass spectrometer. Once prepared for mass spectrometry, samples are generally stable at −20°C for weeks prior to analysis (at least in the absence of the acetonitrile and formic acid).

  Using this approach, the NBD adduct prepared from the thiol form of the protein will exhibit a mass 16 amu less than that prepared from the sulfenic acid form. NBD itself contributes 164 amu to the mass of the protein.

• 10

  Compare the obtained mass with the predicted mass using a computer program that takes into account the natural abundance of all isotopes in the protein.

Materials

• Putative sulfenic acid–containing protein, purified and in a neutral pH buffer (pH 6.5 to 7.5)

• Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 to 7.5 (see APPENDIX 2A)/1 mM EDTA, or other pH 7.0 to 7.5 buffer

• 4 mM 2-nitro-5-thiobenzoic acid (TNB) solution (see recipe)

• 100 mM dithiothreitol (DTT; see recipe)

• Anaerobic quartz cuvette (if required) and regular quartz cuvette

• UV-visible scanning or diode-array spectrophotometer

• Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)

• Additional reagents and equipment for preparing proteins containing sulfenic acid (Support Protocol 1), determining protein concentration by a non-TNB-affected assay (e.g., Detergent-Compatible Protein Assay Kit, Bio-Rad; optional), and performing functional assays (Support Protocol 2; optional)

Prepare protein

• 1

  Prepare at least 1 ml of a 15 μM (monomer concentration) solution of the putative sulfenic acid–containing protein in neutral pH buffer, using the method described in Support Protocol 1 or by other methods established by functional analyses.

  Oxidized proteins should be prepared immediately before use.

Modify protein with TNB

• 2

  To the protein of interest (with or without oxidant or reductant pretreatment as experimental and/or controls samples) add a small volume of 4 mM TNB either directly or through the sidearm of the anaerobic cuvette. Incubate at room temperature and monitor the decrease in A₄₁₂ until the spectral changes are complete (usually within 2 to 5 min, if accessible). Include controls containing peroxide and other oxidants (without protein) to assess the contribution of their reactions.

  Anaerobic conditions may be required to stabilize the sulfenic acid during its generation, as described in Support Protocol 1; if so, this step should also be conducted anaerobically.

  At long incubation times (>20 to 30 min), TNB can air oxidize, a process that must be taken into account using appropriate control reactions.

  A 2- to 3-fold excess of reagent can be used, keeping in mind that the quantitation of sulfenic acids will be most accurate where the total A₄₁₂ of TNB added is 1.0 or slightly less (i.e., within the linear range of the spectrophotometer) and the absorbance change upon adding the reagent to the protein is >0.1.

  Excess peroxide and at least some other oxidants also react with TNB over time; if present, these oxidants should be added to control reactions without protein to assess the contribution of this reaction.

  Denaturation of the protein may be required in this step if the sulfenic acid is not accessible (see the Troubleshooting section in the Commentary).

• 3

  Read the A₄₁₂ of a blank consisting of neutral pH buffer containing the same concentration of TNB as the sample and incubated for the same amount of time.

• 4

  Calculate the sulfenic-acid content of the protein as follows:

  1. Subtract the final A₄₁₂ value of the modified sample (step 2) from the A₄₁₂ of the blank (step 3).

  2. Convert this value to concentration of sulfenic acid–containing residues, using the extinction coefficient 14,150 M⁻¹ cm⁻¹ in buffers of pH 7 or higher (Riddles et al., 1979).

  3. Divide by the protein concentration to obtain the sulfenic acid content of the protein.

     The contribution of excess peroxide and other oxidants that react with TNB over time should be assessed based on the controls.

Isolate TNB-labeled protein

• 5

  Transfer the reaction mixture to an ultrafiltration unit of appropriate molecular weight cutoff (MWCO). Remove unreacted TNB as follows:

  1. Add 6 ml neutral pH buffer.

  2. Concentrate to 50 μl according to manufacturer’s instructions.

  3. Repeat one to three times.

     Attention must be paid to the stated MWCO of the device so that the protein of interest will be retained. With each concentration cycle, the flow-through solution (filtrate) is removed, and fresh neutral pH buffer is added to dilute the protein again, for a total of one to three concentration/redilution cycles.

     Either Centricon ultrafiltration units, which allow 2 ml to be concentrated to 40 to 50 μl, or larger units offered by several other suppliers (e.g., Orbital Biosciences or Vivascience) which allow 6 to 7 ml to be concentrated to ~10 to 50 μl, can be used. In the case of the latter, the ability to concentrate more solution at once and to use swinging bucket rotors, rather than fixed-angle rotors, can speed up the washing process.

• 6

  Check for the presence of free TNB in the filtrate by determining the absorbance at 412 nm. Repeat step 5 until no free reagent is detected (i.e., A₄₁₂ is <0.02).

• 7

  Remove the concentrated protein from the ultrafiltraton unit and bring to approximately the original concentration with neutral buffer. Transfer the solution to a quartz cuvette.

• 8

  Optional: Assess the protein concentration using a protein assay that is not affected by the presence of the TNB—e.g., the Detergent-Compatible (DC) Protein Assay Kit from Bio-Rad.

• 9

  Optional: If identification of the labeled peptide is important, perform a tryptic digest and analyze, as described in Support Protocol 3.

Release TNB from the modified protein

• 10

  Add a 10- to 100-fold excess of 100 mM DTT (up to 5 mM final concentration for 45 μM protein), monitoring the spectral changes from 280 to 600 nm until complete (i.e., until no change is detected).

  The increase in A₄₁₂ is used to assess the amount of TNB released from the isolated, modified protein. The calculation is basically the same as in step 4 except that the A₄₁₂ after DTT treatment is higher than before.

• 11

  Optional: If regeneration of the thiol group(s) in the protein is expected to restore or alter the functional properties of the protein, perform an appropriate assay to determine this (see Support Protocol 2), with or without an additional ultrafiltration step (see step 5), depending mostly on the potential for the excess DTT to interfere with the assay.

Materials

• Putative sulfenic acid–containing protein sample, purified and in a neutral pH buffer (pH 6.5 to 7.5)

• Neutral pH buffer: 25 mM potassium phosphate buffer, pH 7.0 (see APPENDIX 2A)/ 1 mM EDTA, or other pH 7 buffer

• 100 mM 5,5-dimethyl-1,3-cyclohexanedione (dimedone): prepared by adding 14 mg dimedone (Sigma-Aldrich) to 1 ml DMSO; store in aliquots up to several months at −20°C

• Acetonitrile

• Formic acid

• Anaerobic cuvette

• Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)

• Electrospray ionization mass spectrometer (ESI-MS) or access to a fee-for-service facility

• Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)

• Additional reagents and equipment for preparing proteins containing sulfenic acid (Support Protocol 1) and determining the identity of the labeled peptide (Support Protocol 3; optional)

Prepare protein

• 1

  Prepare at least 200 μl of a 15 μM (monomer concentration) solution of the putative sulfenic acid–containing protein in neutral pH buffer, using the method described in Support Protocol 1 or by other methods established by functional analyses.

  Oxidized proteins should be prepared immediately before use.

Modify with dimedone

• 2

  To the protein of interest (with or without oxidant or reductant pretreatment as experimental and/or controls samples) add at least a 100-fold excess (or up to 1 mM) of 100 mM dimedone either directly or through the sidearm of the anaerobic cuvette. Incubate 1 to 2 hr or overnight at 25°C.

  Anaerobic conditions maybe required to stabilize the sulfenic acid during its generation, as described in Support Protocol 1; if so, this step should also be conducted anaerobically.

  Depending on accessibility of the sulfenic acid, denaturation of the protein may be required to promote the labeling reaction. This can be accomplished by adding urea or guanidine hydrochloride to the dimedone, preferably under anaerobic conditions, as denaturation will decrease the stability of the sulfenic acid.

Prepare modified protein for ESI-MS

• 3

  Transfer the reaction mixture to an ultrafiltration unit of appropriate molecular weight cutoff (MWCO). Remove the unreacted dimedone and buffer components as follows:

  1. Add 6 ml deionized water.

  2. Concentrate to 50 μl according to manufacturer’s instructions.

  3. Repeat at least three times.

     Attention must be paid to the stated MWCO of the device so that the protein of interest will be retained. With each concentration cycle, the flow-through solution (filtrate) is removed, and deionized water is added to dilute the protein again, for a total of at least three concentration/redilution cycles.

     Either Centricon ultrafiltration units, which allow 2 ml to be concentrated to 40 to 50 μl, or larger units offered by several other suppliers (e.g., Orbital Biosciences or Vivascience), which allow 6 to 7 ml to be concentrated to ~10 to 50 μl, can be used. In the case of the latter, the ability to concentrate more solution at once and to use swing-out bucket rotors, rather than fixed-angle rotors, can speed up the washing process.

• 4

  Prepare 1 nmol modified protein in 100 μl water. Add an equal volume of acetonitrile and 2 μl formic acid before injection. Analyze by ESI-MS

  This amount of sample is far above that required for the analysis by most modern ESI-MS instruments. Optimization of sample analyses may involve changing the acetonitrile concentration and/or using a volatile buffer such as 10 or 50 mM ammonium bicarbonate instead of water during the washing step (Fuangthong and Helmann, 2002).

  Alternatively, the modified protein can be isolated (without ultrafiltration) by HPLC in tandem with injection into the mass spectrometer, skipping step 3 and the sample preparation above. Once prepared for mass spectrometry, samples are generally stable at −20°C for weeks prior to analysis (at least in the absence of the acetonitrile and formic acid).

  Using this approach, only peptides containing a cysteine sulfenic acid will form adducts with dimedone. Dimedone itself contributes an additional 138 amu to the mass of the protein.

• 5

  Optional: If identification of the labeled peptide is important, perform a tryptic digest of the isolated modified protein and analyze, as described in Support Protocol 3.

Materials

• Putative sulfenic acid–containing protein sample, purified and in a neutral pH buffer (pH 6.5 to 7.5)

• Neutral pH buffer: 25 mM potassium phosphate buffer, pH 6.5 to 7.5 (see APPENDIX 2A)/1 mM EDTA, or other pH 7 buffer

• 250 mM DCP-based affinity and fluorescent reagents (see recipe; see Fig. 17.2.3 and Poole et al., 2007 for structures):

  • Biotinylated DCP-linked probe (DCP-Bio1, DCP-Bio2, DCP-Bio3)

  • Fluorescent-labeled DCP-linked probe (DCP-FL1, DCP-FL2, DCP-Rho1, DCP-Rho2)

• 50 mM and 0.1 M ammonium bicarbonate buffer (not pH adjusted)

• Acetonitrile

• Formic acid

• Acetone or trichloroacetic acid (optional)

• Avidin-conjugated (e.g., streptavidin, monoavidin, or neutravidin) Sepharose or agarose beads

• Phosphate-buffered saline (APPENDIX 2A)

• SDS sample buffer (containing 2-mercaptoethanol; e.g., see APPENDIX 3F)

• Anaerobic cuvette or pear-shaped flask

• Ultrafiltration unit of appropriate MWCO (e.g., Centricon, Millipore; Apollo, Orbital Biosciences; Vivaspin, Vivascience)

• Electrospray ionization mass spectrometer (ESI-MS) or access to a fee-for-service facility

• PD-10 columns (GE Healthcare) or Bio-Gel P6 spin columns (Bio-Rad), optional Software for comparing observed and predicted mass (e.g., Calculate pI/MW tool; http://www.expasy.ch)

• Gel documentation system with filters for fluorescein or rhodamine B

• Additional reagents and materials for preparing proteins containing sulfenic acid (Support Protocol 1), determining the identity of the labeled peptide (optional; Support Protocol 3), purifying proteins (Bollag and Edelstein, 1991), performing SDS-PAGE (APPENDIX 3F or Bollag and Edelstein, 1991), and performing immunoblotting (see UNIT 2.3)

Prepare protein

• 1

  Prepare at least 200 μl of a 15 μM (monomer concentration) solution of the putative sulfenic acid–containing protein in neutral pH buffer, using the method described in Support Protocol 1 or by other methods established by functional analyses.

  Oxidized proteins should be prepared immediately before use.

Modify with DCP-based probe

• 2

  To the protein of interest (with or without oxidant or reductant pretreatment as experimental and/or controls samples) add at least a 100-fold molar excess (or up to 5 mM) of a 250 mM stock of the probe (in DMSO) either directly or through the sidearm of the anaerobic cuvette. Incubate 1 to 2 hr or overnight at 25°C.

  Anaerobic conditions maybe required to stabilize the sulfenic acid during its generation, as described in Support Protocol 1; if so, this step should also be conducted anaerobically.

  Depending on accessibility, denaturation of the protein may be required to promote the labeling reaction. Denaturation of the protein may be required in this step if the sulfenic acid is not accessible (see the Troubleshooting section in the Commentary).

  Some proteins may be sensitive to and precipitate out of solution in the presence of 1% to 2% DMSO, so lower concentrations of the probe stock should be used in those cases.

• 3

  Optional: If identification of the labeled peptide is important, perform a tryptic digest of the isolated modified protein and analyze, as described in Support Protocol 3.

Prepare modified protein for ESI-MS

• 4a

  Transfer the reaction mixture to an ultrafiltration unit of appropriate molecular weight cutoff (MWCO). Remove the unreacted reagent and buffer components as follows:

  1. Add 6 ml deionized water or volatile buffer (e.g., 10 M ammonium bicarbonate).

  2. Concentrate to 50 μl according to the manufacturer’s instructions.

  3. Repeat three to four times (or more).

     Attention must be paid to the stated MWCO of the device so that the protein of interest will be retained. With each concentration cycle, the flow-through solution (filtrate) is removed, and deionized water or buffer is added to dilute the protein again, for a total of at least three concentration/redilution cycles. More washes may be required for sticky fluorescein- and rhodamine-linked reagents.

     Either Centricon ultrafiltration units, which allow 2 ml to be concentrated to 40 to 50 μl, or larger units offered by several other suppliers (e.g., Orbital Biosciences or Vivascience), which allow 6 to 7 ml to be concentrated to ~10 to 50 μl, can be used. In the case of the latter, the ability to concentrate more solution at once and to use swing-out bucket rotors, rather than fixed-angle rotors, can speed up the washing process.

• 5a

  Prepare 1 nmol modified protein in 100 μl water. Add an equal volume of acetonitrile and 2 μl formic acid before injection. Analyze by ESI-MS.

  This amount of sample is far above that required for the analysis by most modern ESI-MS instruments. Optimization of sample analyses may involve changing the acetonitrile concentration and/or using a volatile buffer such as 10 or 50 mM ammonium bicarbonate instead of water during the washing step (Fuangthong and Helmann, 2002).

  Alternatively, the modified protein (without ultrafiltration) can be isolated by HPLC in tandem with injection into a mass spectrometer, skipping steps 4a and 5a. Once prepared for mass spectrometry, samples are generally stable at −20°C for weeks prior to analysis (at least in the absence of the acetonitrile and formic acid).

  Using this approach, only peptides containing a cysteine sulfenic acid will form adducts with the dimedone derivatives (Poole et al., 2005). Each of the derivatives contributes a unique additional mass to the protein (see Poole et al., 2007).

Affinity capture and detect biotin-labeled proteins

• 4b

  Remove excess biotin-containing reagents from the protein sample by ultrafiltration as in step 4a, by gel filtration with PD-10 columns from GE Healthcare or Bio-Gel P6 spin columns to remove small molecules, or by precipitation of proteins with acetone or TCA by standard methods (see Bollag and Edelstein, 1991).

  These methods are compatible with the use of lysis buffer to disrupt cells in tissue culture as a first step; in such cases, the DCP-based biotin reagent can be added directly to the lysis buffer to label cellular proteins. For such samples, subsequent steps can be carried out with phosphate-buffered saline or ammonium bicarbonate buffers.

  Affinity capture of biotinylated peptides may also be performed instead of or in addition to affinity capture of the intact proteins, following proteolytic digestion of the protein sample as described in Support Protocol 3.

• 5b

  Incubate the sample with commercially available avidin-conjugated Sepharose or agarose beads (amounts recommended by manufacturer) for 0.5 to 2 hr (or overnight) at room temperature or 4°C.

  To control for nonspecific binding of “sticky” proteins (e.g., where cellular extracts are being used), samples can be pretreated with chemically similar beads without the avidin (e.g., Sepharose CL-4B beads) prior to application of the sample to the avidin beads.

• 6b

  Wash the beads sequentially two times each with column volumes of:

  • Buffer (e.g., 0.1 M ammonium bicarbonate or phosphate-buffered saline)

  • 1 M NaCl

  • Buffer

  • Water.

  Centrifuge between washes 5 min at 5,000 × g, 4°C and remove the supernatant.

  The stringency of the wash step is of great importance for determining the degree of enrichment obtained for biotinylated versus nonbiotinylated proteins. With a low stringency wash procedure (as described above), one may retain more of the biotinylated proteins, as well as more nonspecifically bound proteins or proteins bound in complexes with those that are biotinylated. For intact proteins, higher stringency washing includes successive incubations with 1% SDS (30 min), 4 M urea (30 min), 1 M NaCl (10 min), 0.1 M ammonium bicarbonate buffer, and then water (Vila et al., 2008).

• 7b

  Elute the biotinylated protein from the beads using one or both (sequentially) of the following (see Vila et al., 2008):

  • 70% acetonitrile with 5% formic acid

  • Boiling in SDS sample buffer (for SDS-PAGE).

• 8b

  Separate biotinylated proteins using standard one-dimensional or two-dimensional polyacrylamide gel electrophoresis methods (APPENDIX 3F or Bollag and Edelstein, 1991), followed by electroblotting and immunodetection with anti-biotin antibody (generally described in UNIT 2.3).

  Alternatively, the primary and secondary antibodies typically used in such detection methods can be replaced with commercially available biotin detection reagents like HRP-conjugated streptavidin.

Detect fluorescein- or rhodamine-labeled proteins

• 4c

  Wash fluorescein- or rhodamine-labeled proteins free of the unreacted reagent as described in step 4a.

• 5c

  Evaluate fluorophore incorporation spectroscopically by the absorbance of the labeled protein in pH 7 phosphate buffer at the appropriate wavelength:

  • 493 nm for protein labeled with DCP-FL1 or DCP-FL2 (extinction coefficient ~ 67,000 M⁻¹ cm⁻¹)

  • 570 nm for protein labeled with DCP-Rho1 or DCP-Rho2 (extinction coefficients ~70,000 or 61,000 M⁻¹ cm⁻¹, respectively).

• 6c

  Separate fluorescent proteins using standard one-dimensional or two-dimensional polyacrylamide gel electrophoresis methods (see APPENDIX 3F or Bollag and Edelstein, 1991), first soaking the gels in water or 50 mM ammonium bicarbonate buffer for 30 min to 1 hr.

• 7c

  Detect fluorescence using appropriate settings and filters for fluorescein or rhodamine B using a gel documentation system.

  Such systems can detect these DCP-linked fluorophores at amounts as low as 0.1 to 0.5 pmol (Poole et al., 2007).

  Because these detection methods do not discriminate between covalently linked fluo-rophores and those binding nonspecifically to proteins of interest, specificity must be demonstrated with proper controls (e.g., for the sulfenic acid modification) and/or by demonstrating covalent modification of the protein of interest by mass spectrometry (step 5a, above).

Materials

• Citrate/phosphate buffer (pH from 3 through 7.6)/1 mM EDTA (see recipe)

• Protein, purified and in 10 mM neutral pH buffer (e.g., 10 mM potassium phosphate buffer pH ~7; see APPENDIX 2A)

• UV-visible spectrophotometer or stopped-flow spectrophotometer (recommended) 3- to 5-ml syringes

• Additional reagents and equipment for preparing proteins containing sulfenic acid (Support Protocol 1) and determining the identity of the labeled peptide (optional; Support Protocol 3)

Prepare sample

• 1

  Prepare at least a 150 μM (monomer concentration) solution of the (putative) sulfenic acid–containing protein in 10 mM neutral pH buffer, using the method described in Support Protocol 1 or other methods established by functional analyses.

  Oxidized proteins should be prepared immediately before use.

• 2

  To observe the expected absorbance change and determine the feasibility of this experiment, mix protein samples with an equal volume of high and low pH citrate/phosphate buffers (e.g., pH 4 and 7.6) and record the UV-visible spectral change from 250 to 400 nm.

  The spectral change should be immediate.

  If the near UV absorbance band is lost at low pH, the following experiment is feasible.

  In the case of the sulfenate anion of the C165S mutant of AhpC, maximal absorbance was at 367 nm (Poole and Ellis, 2002).

Conduct rapid mixing (pH jump) experiment

• 3

  Set the monochromator wavelength of the spectrophotometer at the λ_max for the protein sulfenate peak (e.g., 367 nm in the example above).

• 4a

  For a stopped-flow spectrophotometer: Load the sulfenic acid–containing protein into one 5-ml syringe and one of the buffers (i.e., high or low pH) into another. Rapidly mix the protein and buffer solutions and acquire data at the peak wavelength for 10 sec. Repeat ten times or more for each pH point (minimum of eight suggested), controlling for instrument drift by frequently checking the absorbance of buffer alone.

  For accurate results, this data acquisition should be carried out at a minimum of eight different pH values bracketing the pKa.

  Keep the sample volume as low as possible to minimize protein consumption (although protein may be recovered from this nondestructive procedure at pH values where the protein is stable). Any formation of precipitated protein or any conformational changes resulting from the pH change may, however, confound the results. The use of the stopped-flow spectrophotometer allows for the absorbance changes that ensue after the rapid pH change to be measured before any slower pH-dependent denaturation occurs.

• 4b

  For a standard spectrophotometer: Rapidly mix the buffer and protein by adding the phosphate/citrate buffer to the protein in the cuvette, holding a piece of Parafilm tightly over the cuvette with the index finger, and gently inverting the cuvette several times. Measure the absorbance at the peak wavelength as rapidly as possible. Repeat measurements at each pH value at least three times for a minimum of eight different pH values, as above.

• 5
  Plot absorbance versus pH, averaging all data collected at a given pH. If the expected sigmoidal curve is observed, fit data to the following equation:

  $$y=\frac{\left(A\times {10}^x\right)+\left(B\times {10}^{{\text{pK}}_{\mathrm{a}}}\right)}{{10}^x+{10}^{{\text{pK}}_{\mathrm{a}}}}$$

  where A is the absorbance at high pH;

  • B is the absorbance at low pH;

  • y is the absorbance at pH x.

Materials

• Protein solution

• Argon or oxygen-free nitrogen (optional)

• Neutral pH buffer: 25 mM potassium phosphate (pH 7.0)/1 mM EDTA (or other buffer at pH 7 or lower)

• Peroxide of choice, e.g., 8 mM hydrogen peroxide (H₂O₂) or cumene hydroperoxide (see recipes)

• 100 mM dithiothreitol (DTT; see recipe)

• Anaerobic cuvette assembly (Williams et al., 1979) or small pear-shaped flask with stopcock

• 1

  Determine if anaerobic conditions are necessary for the protein solution of interest, by using one of the methods described in Basic Protocols 1 to 4 for detecting the stabilized protein generated under anaerobic conditions, then use the same method under aerobic conditions.

  If the protein sulfenic acid can be detected under anaerobic conditions, but the sulfenic acid content of the protein decreases markedly when using aerobic conditions, then preparation and initial modification steps of the sulfenic acid–containing protein should be conducted anaerobically.

  Preparation of the sulfenic acid form of the target cysteinyl residue(s) on the protein may or may not require anaerobic conditions for stabilization.

• 2

  Optional: If anaerobic conditions are necessary:

  1. Place the protein solution in an anaerobic cuvette assembly or small pear-shaped flask with stopcock.

  2. While gently rocking, flush with argon or nitrogen for 1 to 2 min, followed by gentle vacuum for 1 to 2 min. Repeat for a total of ten to twenty cycles (20 to 30 min total).

• 3

  Add a stoichiometric amount of the peroxide of choice (e.g., 8 mM H₂O₂ or cumene hydroperoxide) to the protein solution.

  Excess peroxide may also be added, although there is a risk of thiol overoxidation beyond the sulfenic acid oxidation state.

  For cysteine-based peroxidases (e.g., NADH peroxidase and peroxiredoxins) and at least one peroxide-sensitive transcriptional regulator (OxyR), this reaction is very fast (≥10⁵ M⁻¹ sec⁻¹ second-order rate constant); however, in general, the reaction is much slower and varies greatly for other protein thiol or thiolate groups. As a result, no clear statement can be made as to the length of time required for the incubation of the protein with the peroxide (this should be treated as a variable). For example, continue treatment for 2 min to 24 hr at 4° or 25°C.

• 4

  Perform a wavelength scan from 250 to 400 nm to determine if any spectral changes due to addition of peroxide are present.

  Any spectral signature can provide both a way to monitor sulfenic acid formation and a way to discriminate between protonated and deprotonated forms of the sulfenic acid for pK_a determination (see Basic Protocol 5). However, for observations of such low-extinction coefficient spectral changes, the protein needs to be at very high concentration (≥80 μM).

Materials

• Modified protein (see Basic Protocols 1, 2, 3, or 4) and unmodified protein (optional)

• 50 mM N-ethylmaleimide (NEM; optional; see recipe)

• 10 M urea or 8 M guanidine hydrochloride (see recipes)

• 100 mM Tris·Cl buffer, pH 8.0 (APPENDIX 2A) or 100 mM HEPES, pH 7.6 (see recipe)

• 100 mM calcium chloride (1.11 g anhydrous CaCl₂ in 100 ml water; store up to several weeks at room temperature)

• TPCK-treated trypsin solution (see recipe)

• Acetic acid

• 10 mM EGTA

• 100 mM DTT

• 95° or 60°C water bath (optional)

• HPLC equipped with a C18 reversed-phase column, solvents for peptide isolation: e.g., 0.1% (v/v) trifluoroacetic acid, 70% (v/v) acetonitrile with 0.08% trifluoroacetic acid

• Electrospray ionization mass spectrometer (ESI-MS) or access to a fee-for-service facility

• Additional reagents and equipment for performing MALDI-TOF mass spectrometry (optional), SDS-PAGE (APPENDIX 3F; optional), or Tris-tricine-PAGE (APPENDIX 3F; optional)

• 1

  Optional: To block additional cysteine thiol groups, add 50 mM NEM to a 100-fold excess and incubate 1 hr at room temperature to alkylate the additional cysteine thiol groups.

• 2

  Prepare a small volume (≤200 μl) containing ~1 mg isolated, modified, blocked (if necessary) protein and 8 M urea or 6 M guanidinium hydrochloride. Optionally, incubate 15 to 20 min at 95°C, or 45 to 60 min at 60°C to ensure denaturation.

  Protein eluted from avidin or antibody-linked beads into acetonitrile and reduced in volume in vacuo can also be used directly for digestion.

• 3

  Once the sample has cooled, add sufficient 100 mM Tris·Cl, pH 8.0 (or if the sample is NBD-labeled, add 100 mM HEPES, pH 7.6) to lower the denaturant concentration to ≤1 M. Add 1/100 vol of 100 mM calcium chloride (1 mM final).

  Alternatively, ammonium bicarbonate buffer (pH 7.4) can be used as the digestion buffer.

  If NBD- or TNB-labeled protein is used, the conditions for trypsin digestion can be pretested to ensure that the label remains covalently attached to the protein (reductants remove both labels and cannot be used, and amine groups can react with and remove the NBD moiety).

• 4

  Add TPCK-treated trypsin to a protease/protein ratio of 1:100 to 1:40 (w/w). Incubate 12 hr at 37°C. Add a second aliquot of TPCK-treated trypsin solution and incubate another 12 hr.

  Shorter incubation times may also suffice.

• 5

  Optional: To monitor the progress of the digestion, remove small aliquots of the reaction mixture and analyze using reversed-phased HPLC, MALDI-TOF mass spectrometry, SDS-PAGE, or Tris-tricine-PAGE.

• 6a

  For storage: Freeze samples at −20°C and store up to 2 weeks.

• 6b

  For immediate use: Inactivate the trypsin by adding sufficient acetic acid to lower the pH to <4, or by adding 10 mM EGTA to chelate calcium.

• 7a

  To analyze NBD- and TNB-labeled proteins (Basic Protocols 1 and 2) by absorbance: Use the following procedure:

  1. To an aliquot of digested peptide, add 1/10 vol of 100 mM DTT.

  2. Separate the digested peptide samples with and without DTT treatment by HPLC, using an appropriate column and gradient.

     For example, use a C18 or C8 column and a linear gradient over 60 to 100 min from 0% to 60% solvent B (70% acetonitrile/0.08% trifluoracetic acid) with the balance being solvent A (0.1% trifluoracetic acid).

     Monitor the absorbance at 347 or 420 nm for NBD-treated samples, or 325 nm for TNB-treated samples. Peptide elution is typically monitored at 215 nm where the peptide bonds absorb strongly and the contributions of solvents A and B are appropriately balanced.

     For further analysis of these peptides, additional purification using a more shallow gradient should be carried out.

  3. Identify labeled peptides by comparing HPLC chromatograms of the tryptic digests with and without DTT treatment.

     DTT treatment will remove the chromophore and will likely shift the retention time of the peptide.

• 7b

  To analyze fluorophore-labeled proteins (Basic Protocol 4) by fluorescence: Perform reversed-phase HPLC (see step 7a, ii) and identify labeled peptides with excitation and emission wavelengths appropriate to the fluorescent tag used.

  For DCP-FL1 and DCP-FL2: λ_ex,max = 493 nm and λ_em,max ~517 nm.

  For DCP-Rho1 and DCP-Rho2: λ_ex,max = 570 nm and λ_em,max ~588 nm.

  See Poole et al. (2007).

  DTT does not reverse the alkylation with these 1,3-cyclohexanedione-based labeling agents.

• 7c

  To analyze labeled samples by ESI-MS: Perform reversed-phase HPLC (step 7a, ii) in tandem with ESI-MS on digests. Identify labeled residues by the added mass.

• 8

  Optional: Repeat steps 1 to 6 with unlabeled protein and analyze by HPLC in tandem with ESI-MS to verify labeling.

  Where only a single cysteine is encoded within that peptide, further localization is unnecessary. Additional analysis by MS-MS using collision-induced fragmentation can also be used to determine the sequence and location of the labeled cysteine. Alternatively, the peptide can be subjected to Edman degradation to determine the position of the blocked cysteine, although its identification may rely only on the absence of an identifiable peak at the position of the modified cysteine.

Citrate/phosphate buffer

• Combine the following:

• 245 ml water

• 9.61 g citric acid (~200 mM final)

• 7.10 g dibasic sodium phosphate (~200 mM Na₂HPO₄ final)

• 93 mg disodium EDTA

• Measure the pH (should be ~3). Slowly drip in a solution of 1 M sodium hydroxide (4.0 g in 100 ml) to raise the pH, and remove ~10-ml aliquots at each desired pH value (about every 0.2 pH units). Store the aliquots up to 1 month at room temperature, but recheck pH of each before each use.

  These solutions allow for mixing of the sulfenic acid–containing protein with strong buffers at various pH values to determine the pK_a of the protein-associated sulfenic acid (Basic Protocol 5).

Cumene hydroperoxide, 8 mM

• Prepare a stock solution containing 20 μl cumene hydroperoxide solution (80% stock from Sigma; store aliquots up to 1 year at −20°C) and 980 μl dimethyl sulfoxide (DMSO). Prepare a 15× dilution of the stock solution in water to give a final concentration of ~8 mM. Prepare fresh daily.

DCP-based affinity and fluorescent reagents, 250 mM

• Synthesize 1,3-cyclohexanedione linked reagents—including three biotin-linked (DCP-Bio1, DCP-Bio2, DCP-Bio3), two fluorescein-linked (DCP-FL1, DCP-FL2), and two rhodamine-linked reagents (DCP-Rho1, DCP-Rho2)—according to published procedures (Poole et al., 2007). Add 5 mg of one of the compounds to DMSO to give a stock concentration of 250 mM (using DMSO volumes of 50.5, 42.0, 39.2, 36.8, 32.1, 28.3, and 25.2 μl for the different compounds listed respectively). Dispense into aliquots and store up to several months or more at −20°C.

  Each reagent has a unique molecular weight (see Table 1 of Poole et al., 2007).

DTNB, 5 mM

• Prepare a 100 mM stock solution by dissolving 0.198 g of 5,5′-dithiobis(2-nitrobenzoic acid) in 5 ml DMSO. Dispense in aliquots and store up to several months at −20°C. Before use, dilute the stock DTNB solution 20-fold into 25 mM phosphate buffer, pH 7 (see APPENDIX 2A).

• To standardize the working solution: Dilute 3 μl in 0.5 ml 25 mM phosphate buffer. While monitoring the A₄₁₂, add 8-μl aliquots of 0.5 mM DTT, pausing between each addition until the spectral changes are complete. When the A₄₁₂ no longer increases with additional DTT, use the maximal A₄₁₂ to calculate the DTNB concentration, using an ε₄₁₂ value of 14,150 M⁻¹ cm⁻¹ for TNB (the solution resulting from the combination of DTNB and DTT) and the fact that 2 moles of TNB are released per mole DTNB (Riddles et al., 1979).

DTT, 100 mM

• Dissolve 154 mg of 1,4-dithio-DL-threitol (DTT) in 10 ml water. Store in aliquots up to 2 weeks at −20°C.

• To standardize the DTT: First perform the standardization of DTNB (see recipe). Calculate the DTT concentration, using the titration breakpoint from a plot of A₄₁₂ versus volume of DTT added to determine the volume of DTT required to titrate the known amount of DTNB.

  DTT has two thiols per molecule; therefore, its concentration is equivalent to the concentration of DTNB with which it reacts.

Guanidine hydrochloride, 8 M

• To ~4 ml water add 7.64 g guanidine hydrochloride (ultrapure; Sigma-Aldrich). Warm the solution and add water to fully dissolve. Bring the volume to 10 ml total. Store up to several months at room temperature in a tightly sealed bottle.

HEPES, 100 mM (pH 7.6)

• To 90 ml of water, add 2.38 g HEPES and dissolve. Add 1 M sodium hydroxide dropwise to bring the pH to 7.6, and then add water to bring the final volume to 100 ml. Store up to several weeks at room temperature.

Hydrogen peroxide, 8 mM

• Dilute ~182 μl of 30% (v/v) H₂O₂ solution (stored at 4°C) into 250 ml water. Prepare fresh daily.

• To standardize hydrogen peroxide: Prepare solutions of 10 mg/ml o-dianisidine in methanol and 1 mg/ml horseradish peroxidase (HRP) in an appropriate buffer—e.g., 25 mM potassium phosphate (pH 7.0)/1 mM EDTA or other buffer at pH 7; store these solutions up to 1 month or more at 4°C in the dark. Add 10 μl of the 10 mg/ml o-dianisidine solution and 2 to 10 μl of the 8 mM (putative) peroxide solution to 0.9 ml 25 mM phosphate buffer, pH 7 (APPENDIX 2A)/0.1% Triton X-100. Bring to 0.99 ml with deionized water. Use this solution to blank a spectrophotometer at 460 nm, then add 10 μl of the 1 mg/ml HRP solution to the cuvette. Monitor the A₄₆₀ change which is complete within a few seconds. Use ε₄₆₀ = 11,300 M⁻¹ cm⁻¹ for oxidized o-dianisidine and dilution factors to compute peroxide concentration. Use three to four different volumes of the hydrogen peroxide solution to be tested. Perform linear regression to determine the change in A₄₆₀ per microliter (the slope of the line) to obtain an accurate concentration: slope/11,300 × cuvette path length in centimeters = millimolar concentration of hydrogen peroxide.

NBD chloride, 100 mM

• To 1 ml DMSO, add 20 mg 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride). Store in aliquots up to several weeks at −20°C, protected from light.

• To determine the concentration of the solution: First, make a 20-fold dilution of the stock in 25 mM phosphate buffer, pH 7 (APPENDIX 2A), and then add 16 μl of this diluted NBD chloride solution to 1.0 ml methanol, using a glass pipet. Measure the A₃₃₆. Use an ε₃₃₆ of 9800 M⁻¹ cm⁻¹ and the dilution factor to calculate the concentration of the stock.

  Use a glass pipet rather than micropipettor to measure out the methanol and avoid leakage.

  NBD chloride is called 4-chloro-7-nitrobenz-2-oxa-1,3-diazole by Molecular Probes. This reagent is also available from other suppliers (e.g., Sigma-Aldrich). Another abbreviation for the compound is Nbf-Cl.

NBD fluoride, 100 mM

• To 100 μl DMSO, add 1.8 mg of 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD fluoride; Molecular Probes). Keep protected from light at room temperature, and use within several hours after it is prepared.

  Do not store at −20°C as for NBD chloride.

• To determine the concentration of the solution: First, make a 20-fold dilution of the stock into 25 mM phosphate buffer, pH 7 (APPENDIX 2A), and then add 16 μl of this diluted NBD fluoride solution to 1.0 ml methanol, using a glass pipet. Measure the A₃₂₈. Use an ε₃₂₈ of 8000 M⁻¹ cm⁻¹ and the dilution factor to calculate the concentration of the stock.

  Use a glass pipet rather than a micropipettor to measure the methanol and avoid leakage.

NEM, 50 mM

• To 1 ml DMSO, add 6.3 mg N-ethylmaleimide (NEM). Dispense into aliquots and store up to a few weeks at −20°C.

• To standardize the exact concentration (if required): First, prepare a 1:20 dilution of 100 mM DTT (see recipe) and then add 6 μl of this dilution to 1 ml of 25 mM phosphate buffer, pH 7 (APPENDIX 2A). Add 14 μl of a 1:20 dilution of the NEM stock. Incubate 30 min at room temperature, and then add 15 μl of 0.25 mM DTNB, diluted from the 5 mM working solution (see recipe).

  The loss of thiol groups corresponds to the amount of NEM added, allowing for calculation of the concentration of the stock solution. Therefore, the concentration of NEM in the final test solution is equal to the A₄₁₂ of the control (without NEM) minus the A₄₁₂ in the presence of NEM divided by the ε₄₁₂ value of 14,150 M⁻¹ sec⁻¹ for TNB.

TNB solution, 16 mM

• To a dark-colored microcentrifuge tube (or a tube wrapped with aluminum foil) containing 0.84 ml of 25 mM phosphate buffer, pH 7 (APPENDIX 2A), add 80 μl of 100 mM stock DTNB (see recipe) and 80 μl of 100 mM DTT (see recipe). The solution will turn dark orange. Store up to 1 week at −20°C.

• To determine the concentration of TNB: Add 7.5 μl of the TNB solution to 0.5 ml phosphate buffer, measure the A₄₁₂, and calculate the concentration using an ε₄₁₂ of 14,150 M⁻¹ cm⁻¹ for TNB (Riddles et al., 1979). Add 1 μl of 100 mM or 2 μl of 5 mM DTNB (see recipe) and observe any increase in A₄₁₂. If there is an increase, calculate the amount of DTNB that should be added to the stock TNB solution to bring it to the maximum A₄₁₂ but not beyond. This amount will be equal to 1/2 the concentration of TNB generated (according to the increased A₄₁₂ divided by the ε₄₁₂ of 14,150 M⁻¹ sec⁻¹), taking into account the relevant dilutions factions and the volume of the TNB stock. Perform the same test using 100 mM DTT instead of DTNB, adding more DTT to the TNB stock solutions if necessary, as calculated for the needed addition above.

  The reagent is properly prepared if addition of neither dithiothreitol nor DTNB leads to an increase in A₄₁₂. This reagent tends to air oxidize and should be tested every few hours for DTNB formation (readjust with additional DTT as needed).

TPCK-treated trypsin solution

• Prepare a stock solution of 1 mg TPCK-treated trypsin (Worthington Biochemical) in 100 μl of 1 mM HCl (8.5 μl concentrated HCl in 100 ml water), or as indicated by the manufacturer. Dispense into aliquots and store up to several months at −70°C, if necessary, but do not subject to multiple freeze/thaw cycles.

  The best grades of trypsin available (e.g., sequencing-grade trypsin from Promega) include modified forms of trypsin that do not undergo self-digestion and are therefore active longer during the digestion (and do not generate contaminating peptides from the trypsin, itself). Immobilized trypsin (Pierce Biotechnology) can also be used so that the digestion can be stopped by centrifugation and removal of the supernatant to a separate tube.

  Note that for digestion of 1 mg of the target protein using a protease/protein ratio of 1:50, 2 μl of the prepared trypsin solution is added.

Urea, 10 M

• To ~4 ml of water add 6.01 g urea (ultrapure; Sigma-Aldrich). Warm the solution and add water to fully dissolve and bring to 10 ml final volume. Store 1 to 2 days at room temperature.

Background Information

The procedures described herein allow for the detection and measurement of sulfenic acids in proteins by chemical means and do not require the specialized approaches of crystallography or NMR. The intrinsic instability of sulfenic acids makes them inherently diffi-cult to work with; mass spectrometry can theoretically be used to demonstrate the single oxygen added to the cysteine and/or protein, but without use of a trapping agent, only the further oxidized sulfinic (R-SO₂H) and/or sulfonic (R-SO₃H) acid species are typically observed. With NBD modification, the sulfenate oxygen is incorporated into the product, and subsequent mass spectrometry allows for its demonstration. With the other covalent modi-fication methods, the oxygen is lost upon reaction, but the presence of the sulfenic acid in the protein is indicated simply by the ability to react with it.

Critical Parameters

Before any of the spectral analyses can be performed, it must be clear that there is enough protein available to give absorbance changes that are sufficient for quantitative analysis (i.e., >0.1). In the case of the dimedone or 1,3-cyclohexadione-based reagent treatments, which do not necessarily rely on spectral analyses, the requirement for protein may be less, as the amount used must be sufficient for identification of the modification by ESI-MS, or gel-based blotting or fluorescence detection methods.

The chemical modification reagents, if used in excess, do not necessarily have to be standardized by spectral titrations with model reagents unless there are problems with the modification or exact amounts are required. However, the oxidant levels used to generate the sulfenic acid in the first place may be quite critical. For addition of a stoichiometric amount of hydrogen peroxide, for example, exact quantitation of the hydrogen peroxide concentration by the HRP assay is an important first step. As has been mentioned above, the TNB reagent is particularly prone to air-oxidation and must be checked for the presence of excess DTNB or DTT every 2 hr or so.

Loss of protein during ultrafiltration can be a major source of error, so it is advantageous to test in advance for the recovery of a given protein with the specific ultrafitration device to be used.

It is helpful, particularly with the NBD modification methods, to know in advance how many accessible thiols are in the protein. This can be done by adding a 10-fold excess of DTNB and observing the rise in A₄₁₂ over time, until completion of the reaction. The amount of TNB released is directly proportional to the number of reactive thiols present in the protein. However, reactivity and accessibility of given protein thiols to particular reagents can be different.

For pK_a determination, the pH values of all phosphate/citrate buffer solutions used should be measured on the day the analysis is performed.

Troubleshooting

Anticipated Results

Time Considerations

For most of the chemical modification procedures, once the reagents are prepared and the protein concentration is determined, the generation of the sulfenic acid and reaction with the modifying agent is complete within < 1 to 2 hr. Ultrafiltration may take 20 min to 3 hr, depending on the particular device and the molecular weight cutoff (lower cutoff, slower filtration). The pK_a determination itself takes about 1 to 3 hr, with preparation and/or pH measurement of the citrate/phosphate buffers requiring 1 to 2 additional hr. Tryptic digests are carried out for 12 to 24 hr, and each HPLC run takes ~90 to 120 min.