An Introduction to the Use of itraq Reagents for Amino Acid Analysis

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1 An Introduction to the Use of itraq Reagents for Amino Acid Analysis Lisa Sapp Clinical Research and Forensic Toxicology Product Manager Mass Spectrometry Systems

2 Amino Acid Analysis Using itraq Reagents Isobaric Tag (Total mass = 145) Reporter Balance AARG Charged Neutral loss (Mass = 114 & 115) (Mass = 30 & 31) Amine specific Reagent adds amu and brings amino acids out of the noisy low mass region Increases ionization efficiency of amino acids Improves retention on reverse phase column Gives strong signature ion in MS/MS 2 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

3 Amino Acid Analysis Using itraq Reagents Label NHS + AA sample NHS--AA sample + Mix NHS--AA sample NHS--AA Int Std Amt sample = Area 115 x Amt IntStd Area NHS--AA Int Std HPLC Sample Int Std MRM AA MW 114 AA MW 115 API Minutes 3 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

4 Conventional method No change Chemist sample derivatization Chemist sample prep Plasma/urine/CSF SSA (incl NorLeu) precipitation Supernatant Add reaction buffer (incl NorVal) Add itraq reagent 115 Reaction at room temp for 30 min Dry & reconstitute with 114 labeled Int Std Inject 2 μl Physiological 45+ AA Standard mix Add reaction buffer Add itraq reagent 114 Reaction at room temp for 30 min Dilute 114 labeled standards ready for spiking The 114 labeled standards supplied in the kit Chemist does not perform these steps 4 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

5 Apricot Designs TPS-24 System Configuration No manual pipetting Standard 96 well plate format Methods for 12, 24 or 48 sample per batch Off-line centrifuging and drying steps Clear on screen instructions Small footprint 5 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

6 Sar Ans uile Physiological AA 45/32 Thr Analysis Car Chromatogram PSer PEtN Tau Asn Ser Hyp Gly Gln EtN Asp XIC of +MRM (37 pairs): 330.1/115.1 amu Expected RT: 2.4 ID: PSer from Sample 1 (115 PStd + alloile) of ACN combined c.wiff... Cit bala Glu His 3MHis Ala Hcit 1MHis Asa GABA Aad baib Pro Arg Hyl Abu Orn Cth Cys Lys uallo-ile uleu unle Val Met Tyr Nva Hcy Ile Leu Nle Phe Trp Max. 2.3e4 cps. High Resolution Separation 7.3e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 Intensity, cps 4.0e4 3.5e4 3.0e4 2.5e Short analysis 2.0e4 1.5e4 1.0e Time, min 6 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

7 Isomeric/Isobaric Amino Acids All isomeric/isobaric AA s resolved well B Sar βala ALA C XIC of +MRM (37 pairs): 132.1/86.1 amu Expected RT: 6.4 ID: alloile/uile/uleu/unle from Sample 1 (115 PStd + alloile) of ACN combin... Max cps. 3 MHis 6.2e4 Ile/Leu/Nle 6.0e4 5.5e4 Allo-Isoleucine Incorporation 5.0e4 1MHis Intensity, cps 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 Unlabeled amino acids uallo-ile /uile /uleu /unle D E F GABA βaib αaba Val Nval Ile Leu Nle 2.0e4 1.5e4 1.0e Time, min Note: Sar is isobaric with isomers βala and ALA. Other are isomers 7 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

8 AA 45: Acetonitrile Gradient or Methanol Gradient 8 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

9 Cliquid Routine Operation: Run Samples 9 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

10 Cliquid Routine Operation: Report Example 10 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

11 Key Features and Benefits AA 45/32Physiological Amino Acid Analysis Kit High throughput, <15 min analysis per sample vs >160 min for conventional method Robust single universal protocol for quantification 45 amino acids in plasma, serum, CSF, urine, whole blood, and dried blood spots. Incorporates Allo-Isoleucine assay Internal standard for each amino acid Reliable quantification Unequivocal amino acid identification Built-in QC amino acids: Norvaline and Norleucine Complete set of itraq reagents, standards and controls provided in the kit 11 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

12 Key Features and Benefits AA 45/32Physiological Amino Acid Analysis Kit No interference due to sample matrix or overlapping amino acids Results correlate with conventional ninhydrin based systems Cliquid Software for Routine Amino Acid Analysis A state of the art software for AAA acquisition and data processing Significantly less sample required, 40 μl vs 250 μl for traditional AAA system. Amendable to 3/16 inch blood spot samples (poster #577) Rigorously tested with large sample set in leading clinical research labs 12 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

13 General Scheme for Multiplexed Amino Acid Analysis PRG + Amino Acid (sample 1) PRG + Amino Acid (sample 1) PRG + Amino Acid (sample 2) PRG + Amino Acid (sample 3) PRG + Amino Acid (standard) Sample Amount Area 114 Amount Area Amount Amount 117 Area NH P Amino E P T Acid I D E (sample 1) NH P Amino E P T Acid I D E (sample 2) Amino Acid (sample 3) NH P E P T I D E (sample 3) Mix Amino Acid (standard) NH P Amino E P T Acid I D E (sample 1) NH P Amino E P T Acid I D E (sample 2) NH P Amino E P T Acid I D E (sample 3) NH P E P T I D E (sample 4) NH P Amino E P T Acid I D E (sample (standard) 4) AA MW>114 MRM AA MW>115 AA MW>116 AA MW> E E E E E E+04 Area 2.0E E E E+04 Area E E E E Area 0.0E E E E+00 Intensity (cps) Sample 1 Intensity (cps) 5.0E E+04 Sample 2 Intensity (cps) 5.0E E+04 Sample 3 Intensity (cps) 5.0E E+04 Standard Time (min) Time (min) Time (min) Time (min) 13 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

14 New Chemistries Build on the success of AAA Extend chemistry to the analysis of other small molecule analytes Enhanced sensitivity Improved limit of detection Simplified fragmentation pattern Steroid analysis High Throughput Maldi-MRM Analysis of Keto-steroids (Poster # 169, Monday) A Novel Derivatization Reagent to Enhance the Detection Characteristics of Keto-steroids Using LC-MS (Poster # 272, Wednesday) Fatty acids and Eicosanoids Quantitative Analysis of Fatty Acids and Eicosanoids in Physiological Samples by LC/MS/MS Using Isotope Coded Tags (Poster # 260, Monday) 14 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

15 Legal Acknowledgments For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of either Life Technologies Corporation, Applied Biosystems/MDS Analytical Technologies or otherwise, their respective owners Applied Biosystems and MDS Inc. Joint Owners. All Rights Reserved. 15 ASMS Applied Biosystems Inc. and MDS Inc. Joint Owners ABOVE & BEYOND

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