MALDI Calibration Kit I. Introduction The MALDI Calibration Kit is a conveniently packaged selection of MALDI matrices and calibration standards (includes high-purity Neg Ion Mode Calibrant for accurate negative ion mass calibrations) qualified for MALDI-MS applications, and is applicable for calibration and testing of MALDI- MS instrumentation. The MALDI matrices are ultra purified matrices that aid in optimizing desorption/ionization of analytes from a target surface for demanding MALDI applications. Each matrix is prepared under strict quality control. The prepackaged calibration standards eliminate the need to purchase and store large quantities of the component calibration reagents, simplifying sample preparation for TOF mass spectrometry. CONTENTS I. INTRODUCTION II. MALDI MATRICES b. Recommended Usage of MALDI Matrices III. CALIBRATION STANDARDS b. Preparation of Calibration Standards c. Calibration Mixture DIOS Low Mass d. Waters Calibration Mix MALDI Reflectron e. ACTH Standard f. Neg Ion Mode Calibrant IV. ORDERING INFORMATION
II. MALDI MATRICES The matrices are conveniently packaged in 1.5 ml tubes (10 mg of matrix pre-weighed in each tube) for ease of use. Tubes are vacuumsealed in foil pouches for convenient storage and protection from light and air. It is recommended that the matrices be stored at 4 C. After opening, the matrix may be used for 1 month if stored in the dark at 4 C. Once reconstituted in appropriate solvents, the matrix solution is stable for 3 days when stored in the dark at 4 C. b. Recommended Usage of MALDI Matrices The following methods are provided as a general guideline for matrix preparation on standard stainless steel targets. The described method only serves as a starting point. Depending on the application, other solvents and/or methods may give improved results. 1. For optimum performance, use solvents of the highest purity commercially available. CH 3 : Acetonitrile (J. T. Baker Cat. No.: 9017-03) C 2 H 5 OH: Ethanol (Aldrich Cat. No.: 45,984-4) TFA: Trifluoroacetic acid (Fisher, Cat. No: O4902-100) O: Deionized (18.2 MW) water To prepare 0.1% TFA aqueous solution, add 30 µl of TFA to 30 ml of deionized water. 2. Each vial contains 10 mg of matrix material. Depending on the application (peptides, proteins, oligonucleotides, oligosaccharides, etc.), prepare matrix solution by adding appropriate solvent(s) into the matrix vial. 3. Vortex the vial for several seconds to ensure that the solid matrix is completely dissolved. 4. Mix equal volumes of sample solution with the prepared matrix solution. 5. Apply 1 µl onto the MALDI target. 6. Dry at room temperature before submitting target for MALDI analysis. Table 1: Recommended Matrix Solvents and Volumes Matrix Suggested Analytes Solvents and Volumes Concentration Alpha-Cyano-4-Hydroxycinnamic Acid (CHCA) Peptides 495 ml: 495 ml: 10 ml C 2 H 5 OH:CH 3 :0.1% TFA 10 mg/ml Sinapinic Acid Proteins 600 ml: 400 ml 0.1% TFA: CH 3 10 mg/ml 2,5-Dihydroxybenzoic Acid (DHB) Oligosaccharides 100 ml: 400 ml O: CH 3 20 mg/ml 3-Hydroxypicolinic Acid (HPA) Oligonucleotides 200 µl: 200 ml O : CH 3 25 mg/ml 2,4,6-Trihydroxyaceto-phenone (THAP) Oligonucleotides 200 µl: 200 ml O: CH 3 25 mg/ml 2
III. CALIBRATION STANDARDS The calibration mixtures are packaged as lyophilized solids in glass vials. The vials are vacuum-sealed in foil pouches to ensure protection from light and air. It is recommended that all products remain in the foil pouch (in the dark) at room temperature until use. Once reconstituted in appropriate solvents, the solutions are stable when stored at 4 C for the length of time indicated in table below. Stability can be extended by freezing samples at -20 C. b. Preparation of Calibration Standards The following methods are provided as a general guideline for preparation of the standards. The described method serves only as a starting point. Depending on your application, one may consider using other solvents and/or methods. Additionally, the selection of matrix should match that of samples to be analyzed. Optimization for specific applications is recommended. Other relative ratios and solvents may be used for reconstitution. In some cases further dilutions of this solution may yield better spectra depending on the instrument and substrate used. Upon applying 1µl of sample mixture to the target, allow the target to completely dry before submitting it to MALDI analysis. Table 2: Calibration Standards Intended Use and Mass Range PN 186002820 186003926 186003973 Product Description Calibration Mix - MALDI Reflectron Calibration Mix - DIOS Low Mass ACTH Standard Neg Ion Mode Calibrant Intended Use Calibration of MALDI TOF for positive ion reflectron mode detection Calibration of MALDI TOF for small molecule detection in positive reflectron mode External calibration of MALDI TOF in reflectron or linear mode Calibration of MALDI TOF for negative ion reflectron mode detection m/z range 600-4000 1-1500 1000-3000 700-4000 Table 3: Calibration Standards Sample Preparation Methods PN 186002820 186003926 186003973 Product Description Calibration Mix - MALDI Reflectron Calibration Mix - DIOS Low Mass ACTH Standard Neg Ion Mode Calibrant Volume Diluent added to lyophilized calibrant (µl)* Mixed 1:1 with matrix 250 Yes Target Plate Stainless Steel target 250 No DIOS-target Yes 25-50 Yes Stainless Steel target Stainless Steel target * It is recommended as a starting point to use a 1:1 mixture of CH 3 : O as the sample diluent. For optimum performance, solvents of the highest purity commercially available are recommended. The following solvents were found to be satisfactory. CH 3 : Acetonitrile (J. T. Baker Cat. No.: 9017-03) O: High purity (18 MW) water c. Calibration Mixture DIOS Low Mass Waters Calibration Mix DIOS Low Mass contains polyethylene glycol (PEG) 200, 600, and 1000 with sodium iodide, rubidium iodide and cesium iodide (NaI, RbI and CsI). This mixture has not been optimized for use in electrospray systems. The following spectrum of the Calibration Mix DIOS Low Mass was processed by baseline subtraction, smoothing, and centroiding in preparation for calibration. Data was obtained on a Waters MALDI micro MX. Note that the Calibration Mix is relatively concentrated and tends to leave a visible, shiny residue on the surface. Adjust your instrument laser power so that the signal does not saturate the detector. 3
Table 4: MassPREP Calibration Standards Storage Table 5: Peak List for Calibration Mix DIOS Low Mass PN Product Description Solution Storage 186002820 Calibration Mix - MALDI Reflectron Calibration Mix - DIOS Low Mass 1 year 1 year 186003926 ACTH Standard 1 week 186003973 Neg Ion Mode Calibrant 1 year at -20 C Figure 1: MALDI MS spectrum of the Calibration Mix DIOS Low Mass Peak List for Calibration Mix DIOS Low Mass The following reference file contains the masses of selected low mass clusters, which are formed by the mixture of sodium, rubidium, and cesium iodide as well as the sodium (and some rubidium) adducts of polyethylene glycol oligomers with chemical formula HO[C 2 H 4 O]nH + Na (where n = 1,2,3,...) between 200 and 1500 Da. Masses were calculated using the following values (where M (x) indicates the mass, M, of the chemical species, x, within the parentheses): M(C) = 12.000000000 M(H) = 1.007825040 M(N) = 14.003074000 M(O) = 15.994914630 M(Na) = 22.989768 M(K) = 38.9637069 M(Rb) = 84.911792 M(I) = 126.90446 M(Cs) = 132.905429 Below m/z 150 the sodium, rubidium, and cesium ions provide the more dominant s. Above m/z 200 the sodiated PEG s provide the more dominant s. 4 Mass Identity Comment Mass Identity Comment 22.989768 Na 84.911792 86.909185 Rb - Rb - isotope 132.905429 Cs sometimes weak 481.2624806 PEG + Na 499.1582898 PEG + Rb 525.2886954 PEG + Na 543.1845046 PEG + Rb 217.1051919 PEG + Na very weak 569.3149102 PEG + Na 261.1314067 PEG + Na weak 587.2107194 PEG + Rb 279.0272159 PEG + Rb weak 613.341125 PEG + Na 305.1576215 PEG + Na main 631.2369342 PEG + Rb 323.0534307 PEG + Rb main 657.3673398 PEG + Na 349.1838362 PEG + Na 675.263149 PEG + Rb 367.0796455 PEG + Rb 701.3935546 PEG + Na 393.210051 PEG + Na 719.2893638 PEG + Rb 411.1058602 PEG + Rb 745.4197694 PEG + Na 437.2362658 PEG + Na 455.132075 PEG + Rb 763.3155786 PEG + Rb Mass Identity Comment Mass Identity Comment 789.4459841 PEG + Na 1027.472867 PEG + Rb 807.3417934 PEG + Rb 1053.603273 PEG + Na 833.4721989 PEG + Na 1071.499082 PEG + Rb 851.3680081 PEG + Rb 1097.629488 PEG + Na 877.4984137 PEG + Na 1115.525297 PEG + Rb 895.3942229 PEG + Rb 1141.655702 PEG + Na 921.5246285 PEG + Na 1159.551512 PEG + Rb 939.4204377 PEG + Rb 1185.681917 PEG + Na 965.5508433 PEG + Na 1203.577726 PEG + Rb 983.4466525 PEG + Rb 1229.708132 PEG + Na 1009.577058 PEG + Na 1247.603941 PEG + Rb
d. Waters Calibration Mix MALDI Reflectron Waters Calibration Mix MALDI Reflectron contains polyethylene glycol (PEG)1000, 2000, and 3000 with sodium iodide (NaI) and provides many uniformly-spaced s. The following spectrum of the Calibration Mix MALDI Reflectron mixed with MALDI Matrix - CHCA was processed by baseline subtraction, smoothing, and centroiding in preparation for calibration. Data was obtained on a Waters MALDI micro MX. Note that the Calibration Mix is relatively concentrated and tends to leave a visible, shiny residue on the surface. Adjust your instrument laser power so that the signal does not saturate the detector. Figure 2: MALDI MS Spectrum of the Calibration Mix MALDI Reflectron Peak List for Calibration Mix MALDI Reflectron The following reference file contains the masses of the sodium adducts of polyethylene glycol oligomers with chemical formula HO[C 2 H 4 O]nH + Na (where n = 1,2,3,...) between 565 and 4005 Da. Masses were calculated using the following values (where M (x) indicates the mass, M, of the chemical species, x, within the parentheses): M(H) = 1.007825040 M(O) = 15.994914630 M(C) = 12.000000000 M(Na) = 22.989768000 Table 6: Peak List for Calibration Mix MALDI Reflectron Sodiated PEG = M(HO[C 2 H 4 O]nH + Na) n Sodiated PEG = 2286.337287 51 569.3149102 12 2330.363502 52 613.341125 13 2374.389717 53 657.3673398 14 2418.415931 54 701.3935546 15 2462.442146 55 745.4197694 16 2506.468361 56 789.4459841 17 2550.494576 57 833.4721989 18 2594.520791 58 877.4984137 19 2638.547005 59 921.5246285 20 2682.57322 60 965.5508433 21 2726.599435 61 1009.577058 22 2770.62565 62 1053.603273 23 2814.651864 63 1097.629488 24 2858.678079 64 1141.655702 25 2902.704294 65 1185.681917 26 2946.730509 66 1229.708132 27 2990.756724 67 1273.734347 28 3034.782938 68 1317.760562 29 3078.809153 69 1361.786776 30 3122.835368 70 1405.812991 31 3166.861583 71 1449.839206 32 3210.887798 72 1493.865421 33 3254.914012 73 1537.891636 34 3298.940227 74 1581.91785 35 3342.966442 75 1625.944065 36 3386.992657 76 1669.97028 37 3431.018872 77 1713.996495 38 3475.045086 78 1758.02271 39 3519.071301 79 1802.048924 40 3563.097516 80 1846.075139 41 3607.123731 81 1890.101354 42 3651.149945 82 1934.127569 43 3695.17616 83 1978.153783 44 3739.202375 84 2022.179998 45 3783.22859 85 2066.206213 46 3827.254805 86 2110.232428 47 3871.281019 87 2154.258643 48 3915.307234 88 2198.284857 49 3959.333449 89 2242.311072 50 4003.359664 90 5
e. ACTH Standard Waters ACTH standard is Adrenocorticotropic Hormone Fragment 18-39 human, and provides a monoisotopic calibration at 2465.199 (M+H) +. Each vial contains 1nmol of lyophilized ACTH. The spectrum of the ACTH Standard seen below was processed by baseline subtraction and smoothing. The matrix used was CHCA (10mg/mL in 0.1% TFA/90% A/9.9% O). Data was obtained on a Waters MALDI micro MX. Peak List for Neg Ion Mode Calibrant The following reference file contains the masses of the 2-AA Dextran between 789m/z and 4027m/z. Table 7: Peak List for Neg Ion Mode Calibrant Number of hexose, n= M-H (m/z) Number of hexose, n= M-H (m/z) 4 786.26684 15 2568.84764 5 948.31964 16 2730.90044 6 1110.37244 17 2892.95324 7 1272.42524 18 3055.00604 8 1434.47804 19 3217.05884 9 1596.53084 20 3379.11164 10 1758.58364 21 3541.16444 11 1920.63644 22 3703.21724 12 2082.68924 23 3865.27004 13 2244.74204 24 4027.32284 14 2406.79484 Figure 3: MALDI MS Spectrum of the ACTH Standard IV. ORDERING INFORMATION f. Neg Ion Mode Calibrant Waters Neg Ion Mode Calibrant provides polymeric s of 2-AA Dextran. The following spectrum was acquired on a Waters MALDI micro MX using the recommended procedure, including DHB matrix (typically 20-40 mg/ml of DHB in solution with high organic content and 5-10 mm ammonium citrate). Adjust the laser power by aiming the laser at a variety of positions until adequate ionization is achieved. Table 8: MALDI Calibration Kit Product Description Part Number m/z range Units/kit Amount/Unit MALDI Calibration Kit MALDI Matrix CHCA MALDI Matrix DHB ACTH 186003931 Neg Ion Mode Calibrant Calibration Mix R Calibration Mix Low Mass 186003927 -- 1 -- 186002331 -- 5 10 mg 186002333 -- 5 10 mg 186003973 1000-3000 700-4000 600-4000 1 1 nmol 1 50 µg 1 -- 186002820 1-1500 1 -- Figure 4: MALDI MS Spectrum of the Neg Ion Mode Calibrant 6
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