Operating Instructions

Transcription

1 Sequazyme C-Peptide Sequencing Kit Operating Instructions 1 Product Description The Sequazyme C-Peptide Sequencing Kit enables peptide digestion and the analysis of sequentially truncated peptides using mass spectrometry. The Sequazyme C-Peptide Sequencing Kit offers these unique features: Concentration-dependent digestion Peptide digestion is performed directly on the sample plate, using different concentrations of enzyme. This technique minimizes sample handling, sample loss, and method development time. MALDI-TOF mass analysis Masses of truncated peptides, or ladders, are determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The sequence from the C-terminus can be determined from the mass differences between adjacent peaks in the mass spectrum. Note: This kit has been optimized for a Voyager Biospectrometry Workstation. However, it can be used with other mass spectrometers. This technique is fast and provides data that is easier to interpret than data obtained with traditional methods of analysis. With the Sequazyme C-Peptide Sequencing Kit, you can perform a complete sequencing experiment (using a maximum of only a few picomoles of total peptide) in less than 30 minutes. Peptides of the highest purity possible are recommended at concentrations in the range of 100 fmol/µl to 5 pmol/µl to ensure good signal intensities and digestion rates. Mixtures of peptides, such as crude endoprotease digests, can be sequenced. However, mixtures are more difficult to interpret than pure peptides due to overlapping series of peaks from different ladder sequences. 2 Materials Materials provided The Sequazyme C-Peptide Sequencing Kit contains: Buffered carboxypeptidase Y (CPY), 1 vial Buffered carboxypeptidase P (CPP), 1 vial Ammonium citrate buffer (30 mm, ph 6.1), 1 vial, 1.25 ml Ammonium citrate buffer (30 mm, ph 4.5), 1 vial, 1.25 ml Matrix (α-cyano-4-hydroxy cinnamic acid), 1 vial Matrix diluent (50% ACN in 0.3% TFA), 1 vial Peptide sequencing standard (50 pmol/µl of ACTH 18 39), 1vial, 25µl Calibration standard (50 pmol/µl each of ACTH and angiotensin I), 1 vial, 25 µl 3 Preparing Reagents See Section 8, Storing the Kit, for storage and stability conditions of prepared reagents. CAUTION. CHEMICAL HAZARD. Carboxypeptidase Y may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CAUTION. CHEMICAL HAZARD. Carboxypeptidase P may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CPY and CPP specificity Carboxypeptidase Y (CPY) and carboxypeptidase P (CPP) sequentially hydrolyze the C-terminal residues of peptides. CPY has been reported to cleave all residues from the C-terminus, including proline. Certain combinations of residues can inhibit digestion. We have observed that a CPY specificity to residues at the penultimate position (second residue from the end of the fragment) can inhibit the digestion. Inhibition of digestion can often be overcome by shifting the ph of the digestion. See Section 5, Adjusting Digestion ph, for more information. CPP is provided to supplement the information derived from CPY digestions. CPP may be useful to sequence unknown samples that are resistant to CPY. Section Page 1 Product Description Materials Preparing Reagents Sequencing Adjusting Digestion ph Peptide Sequencing Standard Spectra Troubleshooting Storing the Kit Accessories, Spare Parts, and Ordering Information Technical Support

2 Important. Ammonium citrate buffers. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. CAUTION. CHEMICAL HAZARD. Alpha-cyano-4-hydroxycinnamic acid (CHCA) may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING. CHEMICAL HAZARD. Diluent (with acetonitrile) is a flammable liquid and vapor. It may cause eye, skin, and respiratory tract irritation, central nervous system depression, and heart, liver, and kidney damage. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING. CHEMICAL HAZARD. Peptide sequencing standard ACTH may cause an allergic skin and respiratory reaction. Exposure may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. WARNING. CHEMICAL HAZARD. Calibration standard ACTH 18 39/angiotensin I may cause an allergic skin and respiratory reaction. Exposure may cause eye, skin, and respiratory tract irritation. Please read the MSDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Peptide sequencing standard Combine 1 µl of the stock peptide sequencing standard with 49 µl of HPLC-grade water to make a 1 pmol/µl peptide sequencing solution. Vortex to mix. Calibration standard Combine 1 µl of the stock calibration standard with 49 µl of HPLC-grade water to make a 1 pmol/µl calibration solution. Vortex to mix. CPY and CPP 1. Prepare the CPY stock concentrate (Dilution 1) by reconstituting the CPY vial with 150 µl of HPLC-grade water. 2. Prepare the CPP stock concentrate (Dilution 1) by reconstituting the CPP vial with 150 µl of HPLC-grade water. 3. Prepare the following CPY dilutions in clean, 0.5-ml tubes using ph 6.1 citrate buffer: Dilution Number Combine the following: * Use ph 6.1 citrate buffer for CPY dilutions 2 50 µl citrate buffer µl Dilution 1. Vortex to mix µl citrate buffer µl Dilution 2. Vortex to mix µl citrate buffer µl Dilution 3. Vortex to mix µl citrate buffer µl Dilution 4. Vortex to mix. See the Certificate of Analysis provided for the initial CPY concentration. CPY dilutions 1 to 5 are buffered at ph 6.1 with 30 mm ammonium citrate. 4. Prepare the following CPP dilutions in clean, 0.5-ml tubes using ph 4.5 citrate buffer: Dilution Number See the Certificate of Analysis provided for the initial CPP concentration. CPP dilutions 1 to 5 are buffered at ph 4.5 with 30 mm ammonium citrate. Matrix Reconstitute the matrix vial with 1 ml of matrix diluent solution and vortex for at least 10 minutes. Some matrix may remain undissolved. 4 Sequencing Sequencing involves: Checking CPY and CPP activity Digesting peptide samples Analyzing masses Interpreting data and assigning residues 4.1 Checking CPY and CPP Activity Before digesting and analyzing unknown samples, digest and mass analyze the peptide standard to check CPY and CPP activity. 1. Spot the following in each of six consecutive wells across one row of the sample plate. You can use a flat or welled style sample plate. Note: For optimum instrument performance, do not use wells on the outer rows and columns of the sample plate. After adding the enzyme dilution to each standard (see below), mix by withdrawing and expelling the solution with the pipet several times. To well Combine the following: * Use ph 4.5 citrate buffer for CPP dilutions µl citrate buffer µl Dilution 1. Vortex to mix µl citrate buffer µl Dilution 2. Vortex to mix µl citrate buffer µl Dilution 3. Vortex to mix µl citrate buffer µl Dilution 4. Vortex to mix. Add Use a clean pipet tip for each and add µl peptide standard (1 pmol/µl) 0.5 µl CPY Dilution µl peptide standard (1 pmol/µl) 0.5 µl CPY Dilution µl peptide standard (1 pmol/µl) 0.5 µl CPY Dilution µl peptide standard (1 pmol/µl) 0.5 µl CPY Dilution µl peptide standard (1 pmol/µl) 0.5 µl CPY Dilution µl peptide standard (1 pmol/µl) 2. Repeat step 1 using CPP and peptide standard, then spot another six consecutive wells. 3. Spot 0.5 µl of the calibration standard (1 pmol/µl) in a clean well. 4. Wait 5 to 10 minutes to allow the reaction mixtures to evaporate to dryness. Note: If the reactions evaporate in less than 5 minutes, see Section 7, Troubleshooting. 2

3 5. Add 0.5 µl of matrix solution to each well (including the calibration standards) and allow to dry. Visually examine the plate to make sure it is dry. 6. Acquire spectra. See Section 4.3, Analyzing Masses. CAUTION: Do not load the sample plate into the Voyager Biospectrometry Workstation before the plate is dry. A wet plate causes the pressure in the sample chamber to rise, which may generate an error code on the instrument. Evaluating the peptide sequencing standard spectra CPY The spectra for the CPY-digested peptide sequencing standard should qualitatively resemble Figure 2 on page 7. Peak ratios and peak appearance will differ, but you should see a continuous sequence of at least 8 carboxy-terminal residues with no gap in sequence, indicating optimal CPY activity and performance. Note: The sequence of the ACTH peptide sequencing standard is H-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-Ser-Ala- Glu-Ala-Phe-Pro-Leu-Glu-Phe-OH. The spectra for the digested peptide sequencing standard (Figure 2 on page 7) should include peaks that allow the determination of the amino acids shown in bold above. If you see fewer than 8 residues, assuming that the digestion proceeded for 5 to 10 minutes, the CPY is losing activity. Use fresh CPY from a new kit. The peptide sequencing standard provides sufficient overlapping information from adjacent digestions. Under normal circumstances, the sequence should not exhibit gaps. If you see a gap, it may indicate that sample spots are not drying at the same rate, and therefore have different reaction times. Perform the digestion again, and carefully observe each well to ensure that sample is beading and not spreading on the surface. If sample is spreading, clean the plate. See Section 4.5, Sequence Gaps, for more information. Note: If the ACTH parent peak (average mass = 2, Da) is not present in the Dilution 5 spectra, digestion progressed too far. Make one more CPY dilution (Dilution 6) using the protocol in Section 3, Preparing Reagents. Check CPY activity again using all six dilutions. CPP The spectra for the CPP-digested peptide sequencing standard should quantitatively resemble Figure 3 on page 8. A sequence gap at the first two C-terminal residues is normal for digestions with this enzyme:substrate combination. CPP is fully active if three carboxy-terminal residues are digested from the ACTH peptide. If you see fewer than three residues, assuming that the digestion proceeded for 5 to 10 minutes, the CPP is losing activity. Use fresh CPP from a new kit. Note: If the ACTH parent peak (average mass = 2, Da) is not present in the Dilution 5 spectra, digestion progressed too far. Make one more CPP dilution (Dilution 6) using the protocol in Section 3, Preparing Reagents. Check CPP activity again using all six dilutions. 4.2 Digesting Peptide Samples 1. Spot the following in each of six consecutive wells across one row of the sample plate. Note: For optimum instrument performance, do not use wells on the outer rows and columns of the sample plate. After adding the CPY to each sample, mix by withdrawing and expelling the solution with the pipet several times. To well Add 2. Repeat step 1 using CPP dilutions 1 through Spot 0.5 µl of the calibration standard in a clean well. 4. Wait 5 to 10 minutes to allow the reactions to evaporate to dryness. Note: If the reactions evaporate in less than 5 minutes, see Section 7, Troubleshooting. 5. Add 0.5 µl of matrix solution to each well (including calibration standard) and allow to dry. Visually examine the plate to make sure it is dry. 6. Acquire spectra. See Section 4.3, Analyzing Masses. CAUTION: Do not load the sample plate into the Voyager Biospectrometry Workstation before the plate is dry. A wet plate causes the pressure in the sample chamber to rise, which may generate an error code on the instrument. 4.3 Analyzing Masses Use a clean pipet tip for each and add µl sample 0.5 µl CPY Dilution µl sample 0.5 µl CPY Dilution µl sample 0.5 µl CPY Dilution µl sample 0.5 µl CPY Dilution µl sample 0.5 µl CPY Dilution µl sample 1. Acquire one spectrum from the calibration standard. Calibrate using the appropriate average or monoisotopic masses of the angiotensin 1 and ACTH peaks: Calibrant Monoisotopic Mass (Da) Average Mass (Da) Angiotensin I 1, , ACTH , , Edit the method to use the calibration file created in step Acquire at least three spectra per well for the digestion series (including the well that does not contain CPY or CPP). Note: Data analysis is based on a statistical treatment. Therefore, multiple replicates from each well are required. More replicate data sets yield more accurate mass assignments. Before acquiring additional replicates, read Determining if you need statistics on page 4. 3

4 4. After acquiring spectra for all wells, calculate peak masses. Set the threshold high enough to detect only the peaks of interest, or manually mark the peaks. See the Voyager Biospectrometry User s Guide. Note: Distinguishing between peaks of interest and chemical or instrument noise may be difficult. You may need to experiment to accurately identify peaks of interest. 4.4 Interpreting Data and Assigning Residues This section describes how to manually assign residues. If you have the DEcode Sequence Analysis software, skip this section and refer to the DEcode Sequence Analysis Software User s Guide for instructions on automatically assigning residues. See Section 9, Accessories, Spare Parts, and Ordering Information for DEcode software ordering information. If you have the Data Explorer software, version 3.5 or later, skip this section and refer to the Data Explorer Software User s Guide for instructions on automatically assigning residues by using the Ladder Sequencing Toolbox. Overview After digestion, data acquisition, and mass calculation (described in previous sections) the mass differences between adjacent peaks in the ladders are used to determine the C-terminal sequence of the peptide. By acquiring multiple spectra, you can assign residues based on a statistical treatment of the replicate data sets. This statistical treatment of data involves: Calculating average mass differences with associated standard deviations for each of the replicates. Using a t-test to determine the statistical likelihood that a given mass difference represents an amino acid. The t-test compares an experimental average mass difference to a known amino acid mass to arrive at a calculated t-value (t calc ). This measure ideally permits rejection (at a very high probability) of all but one possible residue. Determining if you need statistics In most cases, the experimental average mass difference for a pair of peaks is close to only one known residue mass and is not easily confused with any other residues. For example, a mass difference of 70.4 Da most likely represents Ala (MW = Da), because no other residue mass is close to this mass. In general, a well-calibrated, linear, time-of-flight mass spectrometer generates potentially ambiguous data only for the following sets of residues: Masses of Da may be Ile/Leu, Asn, or Asp Masses of Da may be Gln, Lys, or Glu The following statistical analysis of the mass differences in the above ranges should lead you to make a confident residue assignment. Assigning residues statistically 1. Calculate the mass difference between the highest mass pair of adjacent peaks in each spectrum that includes the pair. 2. Calculate the average mass difference and standard deviation for the highest mass pair of adjacent peaks. 3. Perform a t-test for the highest mass pair of adjacent peaks: Refer to Table 1, Masses of Common and Modified Amino Acids (listed in mass order) on page 5, and find the mass closest to the calculated average mass difference. Note the two masses that bracket the mass closest to the calculated average mass difference. Use the equation in Figure 1 to determine the t calc value for the two masses noted above. t calc = x µ n where: s x = average calculated mass difference from step 2 µ = known mass value of residue from Table 1 n = number of replicates of adjacent pairs of peaks s = calculated standard deviation from step 2 Figure 1 t-test Equation 4. Refer to Table 2 on page 5 and note both the t table value for the appropriate number of replicates for the pair of adjacent peaks and the confidence level you need. 5. Compare t calc and t table values: If t calc > t table The residue used in the t-test can be statistically ruled out at the selected level of confidence If t calc < t table The residue used in the t-test cannot be statistically ruled out at the selected level of confidence Note: It is not statistically possible using mass analysis to distinguish between the isomeric pair Leu and Ile. Mass accuracy of your mass analyzer affects your ability to distinguish between residues with similar masses. If t-test results do not satisfactorily rule out enough residues, acquire more replicates of the dilution that contains the pair of adjacent peaks in question. The greater the number of replicates used in the calculation, the more likely you can limit the list of possible residues to a single residue. 6. If you can rule out the masses that bracket the mass closest to the calculated average mass difference, no further calculations are necessary. If you cannot rule out the masses that bracket the mass closest to the calculated average mass difference, repeat step 3 through step 5 for the highest mass pair of adjacent peaks, using the next highest and next lowest masses listed in Table 1, Masses of Common and Modified Amino Acids. 4

5 7. Repeat step 1 through step 6 for all remaining pairs of adjacent peaks. Note: You will have a different number of mass differences to calculate for different pairs of adjacent peaks, because the same adjacent pairs of peaks may or may not appear in more than one dilution. Table 1 Masses of Common and Modified Amino Acids Name Symbol Monoisotopic Mass Average Mass Common Amino Acids glycine Gly alanine Ala serine Ser proline Pro valine Val threonine Thr cysteine Cys leucine/isoleucine Leu/Ile asparagine Asn aspartamine Asp glutamine Gln lysine Lys glutamic acid Glu methionine Met histidine His phenylalanine Phe arginine Arg tyrosine Tyr tryptophan Trp Terminal Groups N-terminal hydrogen H N-terminal formyl Form N-terminal acetyl Ac C-terminal free acid -OH C-terminal amide -NH Common Ionization Adduct Masses 1 proton H sodium Na potassium K Masses represent the difference between the mass of the analyte of interest and the mass of the ionized form. Table 2 Tabulated t table Values 1 Number of Replicates of Pairs of t table Values Confidence Interval Adjacent Peaks 90% 95% 99% See the CRC Handbook of Chemistry and Physics for a more detailed table of values. Example The first adjacent pair of peaks for the peptide sequencing standard ACTH 18 39, shown in Figure 2 on page 7, yields this information: Average Mass (x) Standard Deviation (s) 0.31 Number of Replicates 9 (3 replicates each of Dilutions 3, 4, and 5) Table 3 lists the results of the t-test for the first adjacent pair of peaks (the selected t table values) and compares t calc and t table to determine if residues can be ruled out. Table 3 t-test Values for First Pair of Adjacent Peaks in ACTH Amino Acid t calc 1 t calc >t table (2.306) 2 Can Residue be ruled out? Gly yes Ala yes Ser yes Pro yes Val yes Thr yes Cys yes Ile yes Leu yes Asn yes Asp yes Gln yes Lys yes Glu yes Met yes His yes Phe 1.19 NO Arg yes Tyr yes Trp yes 1 Calculated using x = ; s = 0.31; n = % confidence interval All residues except Phe can be ruled out based on the comparison of t calc and t table. Table 4 contains the residue assignments at a 95% confidence interval and the values used to determine the assignments for ACTH shown in Figure 2 on page 7. 5

6 Table 4 Calculated Values and Residue Assignments for ACTH Adjacent Pair Average Mass (x) 4.5 Sequence Gaps Standard Deviation (s) A sequence gap is a point in a spectrum where the mass difference between an adjacent pair of peaks represents the loss of more than one amino acid. A gap can occur if the rate of hydrolysis at a given point in the sequence is so great that no population of the truncated peptide remains at a detectable level. You can deal with sequence gaps in two ways: Repeat the digestion and adjust the ph of digestion to adjust the rate of hydrolysis and attempt to fill in the gap. See Section 5, Adjusting Digestion ph, for information. Use di- or tri-amino acid masses instead of the masses listed in Table 1 to statistically assign residues. You may be able to determine the amino acid composition of the gap. 5 Adjusting Digestion ph Number of Replicates Residue Phe Glu Leu/Ile Pro Phe Ala Glu Ala 1 95% confidence interval If digestion does not occur with unknown samples, or if you observe gaps in the sequence, you may be able to alter digestion by adjusting the ph. You need TFA (neat) and ammonium hydroxide (30% solution) for this procedure. They are not provided in the kit. Note: If digestion does not occur with the peptide sequencing standard, and you have properly prepared the dilution series using the ammonium citrate buffer provided, there is a problem with the enzyme, not a problem with the ph of the digestion. Perform two digests for each enzyme at approximately 1 ph unit higher and 1 ph unit lower than the starting ph. The starting ph values are: Enzyme Approximate starting ph if your sample is in: Water CPY % TFA If digestion does not occur: 1. Prepare two sample dilution series for CPY and two sample dilution series for CPP, as described in Section 4.2, Digesting Peptide Samples. 2. To the wells in one CPY dilution series, add 0.5 µl of the appropriate percentage of TFA listed below. Mix. 3. To the wells in the other CPY dilution series, add 0.5 µl of the appropriate percentage of ammonium hydroxide listed below. Mix. Note: If your sample is not in water or TFA, account for the buffering capacity and ph of the sample buffer when choosing the percentage of TFA or ammonium hydroxide to use. Adjusting Solution for CPY % Adjusting Solution (sample in water) % Adjusting Solution (sample in 0.1% TFA) TFA Ammonium hydroxide ph of Reaction 1 1 Assumes a 1:1:1 addition of peptide, CPY, and adjusting solution. 4. To the wells in one CPP dilution series, add 0.5 µl of the appropriate percentage of TFA listed below. Mix. 5. To the wells in the other CPP dilution series, add 0.5 µl of the appropriate percentage of ammonium hydroxide listed below. Mix. Note: If your sample is not in water or TFA, account for the buffering capacity and ph of the sample buffer when choosing the percentage of TFA or ammonium hydroxide to use. Adjusting Solution for CPP % Adjusting Solution (sample in water) % Adjusting Solution (sample in 0.1% TFA) TFA Ammonium hydroxide ph of Reaction 1 1 Assumes a 1:1:1 addition of peptide, CPP, and adjusting solution. 6. Allow the reactions to evaporate to dryness. 7. Add 0.5 µl of matrix solution to each well (including calibration standard) and allow to dry. Visually examine the plate to make sure it is dry. 8. Analyze the samples. If digestion is not enabled by adjusting the ph, the C-terminus of the peptide is resistant to CPY or CPP hydrolysis. Increasing the temperature may enable digestion. If it does not, proceed to the next sample. See Section 7, Troubleshooting, for more information. CPP

7 6 Peptide Sequencing Standard Spectra The CPY-digested peptide sequencing standard spectra below were obtained after concentration-dependent digestion of ACTH on a Voyager Biospectrometry Workstation No CPY (well 6) No CPY Dilution 5 (well 5) Dilution Dilution 4 (well 4) Dilution Dilution 3 (well 3) Dilution Dilution 2 (well 2) Dilution Dilution 1 (well 1) Dilution m/z (Da) Ala Glu Ala Phe Pro Leu Glu Phe Figure 2 CPY-Digested Peptide Sequencing Standard Spectra 7

8 The CPP-digested peptide sequencing standard spectra below were obtained after concentration-dependent digestion of ACTH on a Voyager Biospectrometry Workstation No CPP (well 6) Dilution 5 (well 5) Dilution 4 (well 4) Dilution 3 (well 3) Dilution 2 (well 2) Dilution 1 (well 1) m/z (Da) Ala Glu Ala Phe Pro Leu Glu Phe Figure 3 CPP-Digested Peptide Sequencing Standard Spectra 8

9 7 Troubleshooting Symptom Possible Cause Action No ladders in spectrum, parent peak only. Peptide not digested. Poor matrix crystallization CPY or CPP not active Digestion rate of some amino acid sequences is ph-dependent CPY-sample solution or CPP-sample solution evaporates before digestion occurs Check activity of enzyme. See Section 4.1, Checking CPY and CPP Activity. If enzyme is not active, reconstitute fresh enzyme from a new kit and prepare dilution series. Adjust the ph of the digestion. See Section 5, Adjusting Digestion ph. Extend digestion time by doing one of the following: Add 1 to 2 µl of HPLC-grade water to the well to keep CPY or CPP and sample in solution longer. Increase the volume of CPY or CPP and sample used from 0.5 µl to 1.0 µl. Increase ambient humidity during digestion. Peptide is resistant to CPY Increase the temperature of the digestion to 37 C and increase ambient humidity during digestion. Proceed to digestion with CPP. Peptide is resistant to CPP Increase the temperature of the digestion to 37 C and increase ambient humidity during digestion. Proceed to the next sample if increased temperature does not enable digestion. Buffer salt concentration in sample-matrix solution has increased ph above 3. Matrix does not crystallize properly above ph 3. Increase TFA percentage in matrix 0.5% or more to decrease the sample-matrix solution ph below 3. Digestion dries too fast (<5 minutes) Adduct peaks Unidentified peaks in mass spectra Reconstituted matrix frozen before use Matrix has degraded, no longer viable High ambient temperature or low ambient humidity Sample spreads on plate, instead of beading. Plate surface is not clean. Peaks of mass 22 or 38 Da from a parent caused by Na + and K + adducts Peaks of mass 16 Da (or multiples of 16) caused by oxidation of methionine, tryptophan, or both in sample (likely to occur during the digestion on the plate) CPY or CPP degraded Matrix has degraded, no longer viable Freezing degrades matrix. Use matrix stored at 4 C. Prepare fresh matrix using α-cyano-4-hydroxy cinnamic acid at 10 mg/ml in 50% ACN in 0.3% TFA. Add 1 to 2 µl of HPLC-grade water to the well to slow drying time. Clean the plate. See the Voyager Biospectrometry User s Guide. Desalt sample. Increase TFA percentage in matrix 0.5% or more. Ignore the adducts. Perform digestion under a blanket of an inert gas. Ignore peaks. Mass analyze the CPY dilution series or CPP dilution series with no sample. If peaks are present, enzyme has degraded. Reconstitute fresh enzyme from a new kit and prepare dilution series. Prepare fresh matrix using α-cyano-4-hydroxy cinnamic acid at 10 mg/ml in 50% ACN in 0.3% TFA. 9

10 8 Storing the Kit Store the Sequazyme C-Peptide Sequencing Kit and components of the kit under the following conditions: 9 Accessories, Spare Parts, and Ordering Information Item Quantity Part Number Kit Component Store At Stability Unopened kit <0 C 1 year (see expiration date on kit box) Reconstituted CPY or CPP Short-term 4 C 2 weeks Long-term <0 C 4 weeks Note: Do not refreeze the kit more than five times. Freezing more than 5 times reduces enzyme activity by more than two-fold. Peptide sequencing standard Calibration standard Reconstituted matrix <0 C <0 C 1 year 1 year 4 C 1 week Note: Do not freeze the reconstituted matrix. Sequazyme C-Peptide Sequencing Kit Sequazyme C-Peptide Sequencing Kit with DEcode Sequence Analysis Software Kits include: Buffered carboxypeptidase Y (CPY) Buffered carboxypeptidase P (CPP) Ammonium citrate buffer ph 6.1 Ammonium citrate buffer ph 4.5 Matrix (α-cyano-4-hydroxy cinnamic acid) Matrix diluent (50% ACN in 0.3% TFA) Peptide sequencing standard (50 pmol/µl of ACTH 18 39) Calibration standard (50 pmol/µl each of ACTH and angiotensin I) 1 kit P kit P vial 1 vial 1 vial, 1.25 ml 1 vial, 1.25 ml 1 vial 1 vial 1 vial, 25 µl 1 vial, 25 µl 10 Technical Support Applied Biosystems is committed to meeting the needs of your research through enabling technologies like the Sequazyme C-Peptide Sequencing Kit. Our dedicated support staff is available to answer questions about using this product to the fullest extent possible. Applied Biosystems offers a complete line of time-of-flight (TOF) mass spectrometry products to meet your analysis needs. Please contact your Applied Biosystems representative for technical and ordering information. Applied Biosystems publishes a continuing series of Application Notes and Technical Notes, highlighting ladder sequencing applications and strategies. For a publications list, or for further details or answers to questions related to other products, contact Applied Biosystems using the information on the back page. 10

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12 Copyright 2000, 2001, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. Applied Biosystems is a registered trademark, and AB (Design), Applera, Biospectrometry, Data Explorer, DEcode, Delayed Extraction, Sequazyme, and Voyager are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. Headquarters 850 Lincoln Centre Drive Foster City, CA USA Phone: Toll Free (In North America): Fax: Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our web site at All other trademarks are the sole property of their respective owners. Applera Corporation is committed to providing the world s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in the USA, 07/2001 Part Number Rev. B