Bioanalytical Laboratories LC-MS/MS Assay for Amyloid-β Peptides in CSF: Materials and Preparation Procedures R&D Bioanalytical Laboratories 2244 Dabney Road Richmond, VA 23230 GBSC Materials Group 30 Jan 2012

Outline Aβ peptide reference standards Stock solution preparation Intermediate/working solutions preparation Calibrators preparation Artificial cerebrospinal fluid (aCSF) matrix Sample preparation (pretreatment)

Aβ peptide reference standards Analytes AnaSpec: Aβ1-14, Aβ1-15, Aβ1-17, Aβ1-34, Aβ1-38, Aβ1-43 rPeptide: Aβ1-40, Aβ1-42 Phoenix Pharmaceuticals: APL1β28 Alternative sources: American Peptide, Bachem, others? Internal Standards rPeptide: [15N51] Aβ1-38, [15N53] Aβ1-40, [15N55] Aβ1-42 Sponsor: [13C9,15N] Aβ1-15, [13C9,15N] Aβ1-17

Aβ1-42 reference standards Vendor/Catalog #: rPeptide, A-1002; A-1102 [15N] Aβ1-42 Product Name: Beta-Amyloid (1-42), Ultra Pure; Beta-Amyloid (1-42), 15N Uniform Labeled Source: Recombinant. DNA sequence encoding human beta-amyloid (1-42) sequence was expressed in E. coli; [15N] Aβ1-42 expressed with 15N as the nitrogen source Sequence: D A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L M V G G V V I A Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala- Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala Molecular Mass: 4512.7; 4567.3 [15N] Aβ1-42 Peptide Purity: >97% Counter Ion: TFA Supplied As: 1 mg/vial white lyophilized powder Re-suspension: Suggest 1% NH4OH at a concentration of 1 mg/mL, sonicate for 30 seconds to 1 minute after dissolving. To bring it into buffer: After re-suspension, add 5-10x buffer stock, bring to 1x with water. Storage: –20 °C

Stock solution preparation Aβ1-42 Stock, 0.5 mg/mL Reconstitute Aβ1-42 lyophilized powder in original vial with 2.00 mL DMSO, added using a Hamilton Gastight® syringe (Product No. 81317), to a nominal concentration of 0.500 mg/mL. Allow 5.0 min for equilibration, then vortex for 1.0 minute at medium speed, equilibrate solution at room temperature for 30 min, then vortex for another 1.0 minute at medium speed. Sub-aliquot stock solution (100 μL each) into 0.5-mL Eppendorf Protein LoBind microcentrifuge tubes (Product No. 022431064), store at -80 C.

Intermediate solution preparation Intermediate Combined Solution, 1000/2000/4000 ng/mL Combine appropriate volumes of individual analyte stocks in DMSO into a 12.0-mL Nunc-Immuno Minisorp tube (Product No. 12-565-148) to obtain nominal concentrations of 1000 ng/mL for Aβ1-14 and -15, 2000 ng/mL for Aβ1-17, -34, -38, -42, and -43, and 4000 ng/mL for Aβ1-40 and APL 1β-28. Allow 5.0 min for equilibration, vortex for 0.5 minute at medium speed, equilibrate at room temp for 30 min, then vortex for another 0.5 minute. Sub-aliquot solution (500 μL) into 0.5-mL Eppendorf LoBind microcentrifuge tubes and store at -80 C.

Intermediate (IS) solution preparation Intermediate Combined Internal Standard Solution, 500 ng/mL Combine appropriate volumes of individual IS stocks in DMSO into a 12.0-mL Nunc-Immuno Minisorp tube to obtain a nominal concentration of 500 ng/mL for all internal standards. Allow 5.0 min for equilibration, vortex for 0.5 minute at medium speed, equilibrate at room temp for 30 min, then vortex for another 0.5 minute. Sub-aliquot solution (500 μL) into 0.5-mL Eppendorf LoBind microcentrifuge tubes and store at -80 C.

Working (IS) solution preparation Working Internal Standard (WIS) Solution, 40.0 ng/mL Prepare WIS solution in artificial CSF containing 5% rat plasma in 2.0-mL Eppendorf LoBind microcentrifuge tubes at a nominal concentration of 40.0 ng/mL for all internal standards. Allow 5.0 min for equilibration, vortex for 0.5 minute at medium speed, equilibrate at room temp for 30 min, then vortex for another 0.5 minute. Prepare fresh daily or store at -80 C.

Calibrator working solutions preparation Calibrator Working Solutions, 625 to 250,000 ng/mL (25x) Prepare calibrator working solutions in DMSO in 12.0-mL Nunc-Immuno Minisorp tubes at 625 to 250,000 ng/mL, depending on the analyte (25x the final calibration standard concentrations). Allow 5.0 min for equilibration, vortex for 0.5 minute at medium speed, equilibrate at room temp for 30 min, then vortex for another 0.5 minute. Sub-aliquot each working standard solution (40 μL) into 0.5-mL Eppendorf LoBind microcentrifuge tubes for future calibration standards preparations and store at -80 C.

Calibration standards preparation Calibration Standards, 25.0 to 10,000 ng/mL Add 0.960 mL artificial CSF to 2.0-mL Calibrator Working Solution tubes (containing 40-μL CWS aliquots) to prepare calibrators at nominal concentrations of 25.0 to 10,000 ng/mL, depending on the analyte. Allow 5.0 min for equilibration, vortex for 0.5 minute at medium speed, equilibrate at room temp for 30 min, then vortex for another 0.5 minute. Prepare fresh daily.

Artificial CSF Preparation Alzet Solution A: 17.32 g of sodium chloride (NaCl) 0.448 g of potassium chloride (KCl) 0.412 g of calcium chloride dihydrate (CaCl2 ・ 2H2O) 0.326 g of magnesium chloride hexahydrate (MgCl2 ・ 6H2O) Dissolve in 1.0 L water and store at 2 to 8 C for up to 3 months. Modified Alzet Solution B: 0.428 g of sodium phosphate dibasic heptahydrate (Na2HPO4 ・ 7H2O) 0.054 g of sodium phosphate monobasic monohydrate (NaH2PO4 ・ H2O) 0.5 g of human serum albumin 0.05 g of human IgG 1.6 g of glucose Dissolve in 1.0 L water, store at -20 C for up to 3 months. Avoid freeze-thaw cycles..

Artificial CSF preparation (cont.) Artificial Cerebrospinal Fluid (aCSF): 1:1, Alzet Solution A / Modified Alzet Solution B, v/v. Combine 500 mL of Alzet solution A and 500 mL of modified Alzet solution B. Adjust the pH to 7.3 with either dilute HCl or dilute NaOH as needed. Store at –20 C for up to three months. Avoid repeated freeze-thaw cycles. Note: aCSF protein: 0.275 g/L, normal CSF protein: 0.15-0.45 g/L Alzet reference: (http://www.alzet.com/products/guide_to_use/CFS_preparation.html)

Sample preparation (pretreatment) Aliquot 200 µL CSF or aCSF sample. Add 20 µL of 4.0 mg/mL BSA in PBS. Vortex at 600 RPM for 5 minutes. (Should additional BSA vs. rat plasma be incorporated into aCSF only?) Add 10 µL of 40 ng/mL Working Internal Standard Solution. Vortex at 600 RPM for 5 minutes. Add 200 µL of 5 M guanidine hydrochloride. Vortex at 600 RPM for 45 minutes at room temperature. Add 200 µL of 4% (v/v) phosphoric acid. Vortex at 600 RPM for 2 minutes. Extract samples using Waters Oasis MCX μElution SPE procedure (originally developed by Erin Chambers).