[ Care and Use Manual ]

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1 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

2 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.: ) C 2 H 5 OH: Ethanol (Aldrich Cat. No.: 45,984-4) TFA: Trifluoroacetic acid (Fisher, Cat. No: O ) 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

3 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 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 Table 3: Calibration Standards Sample Preparation Methods PN 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 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.: ) 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

4 Table 4: MassPREP Calibration Standards Storage Table 5: Peak List for Calibration Mix DIOS Low Mass PN Product Description Solution Storage Calibration Mix - MALDI Reflectron Calibration Mix - DIOS Low Mass 1 year 1 year ACTH Standard 1 week 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) = M(H) = M(N) = M(O) = M(Na) = M(K) = M(Rb) = M(I) = M(Cs) = 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 Na Rb - Rb - isotope Cs sometimes weak PEG + Na PEG + Rb PEG + Na PEG + Rb PEG + Na very weak PEG + Na PEG + Na weak PEG + Rb PEG + Rb weak PEG + Na PEG + Na main PEG + Rb PEG + Rb main PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb Mass Identity Comment Mass Identity Comment PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb PEG + Rb PEG + Na PEG + Na PEG + Rb

5 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) = M(O) = M(C) = M(Na) = Table 6: Peak List for Calibration Mix MALDI Reflectron Sodiated PEG = M(HO[C 2 H 4 O]nH + Na) n Sodiated PEG =

6 e. ACTH Standard Waters ACTH standard is Adrenocorticotropic Hormone Fragment human, and provides a monoisotopic calibration at (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) 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 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 Neg Ion Mode Calibrant Calibration Mix R Calibration Mix Low Mass mg mg nmol 1 50 µg Figure 4: MALDI MS Spectrum of the Neg Ion Mode Calibrant 6

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