AccQ•Tag interfering withtheinterpretation ofresults.Insome interfering peaks tothestandard chromatogram,potentially modified proteinsamplesoftenproduce additional In aminoacidanalysis,constituentsofnon- or AccQ•Tag™ Method Retention TimeTables forAminoAcidsUsingthe rpohn51.034 1.000 0.987 0.980 0.960 0.961 0.913 0.942 5 0.884 1 7 0.843 8 1 0.840 hydrolysate method(eluent pH 5.05). 7 0.825 1 This tableindicates theRC’s ofthe derivatives usingthestandard 1 0.791 0.781 Tryptophan 6 Phenylalanine 0.825 8 Nitrotyrosine 1 1 Methyllysine 1 Leucine 0.695 1 0.757 Isoleucine 0.708 Lysine 3 0.737 0.693 Methionine 0.656 0.664 Cysteine, Thiobutyric 1 3 7 Tyrosine 3 0.660 0.662 Cysteine, Propyltriethyl 0.646 7 Cysteine, Thiopropionic 0.637 1 0.649 7 0.628 α Methylarginine-2 0.640 γ 0.640 0.618 3 3 Dimethylarginine 3 Methylarginine-1 1 3 1 0.610 Cysteine, Propyltrimethyl 0.626 Methionine Sulphone 0.621 3 0.605 3 Alanine 0.566 5 0.586 0.519 Cysteine, Thioglycolic 0.575 Phosphotyrosine 0.518 0.501 Cysteine, Ethyltrimethyl 7 Sulfopropyl Cysteine 3 Threonine 3 6 0.446 1 6 1 7 0.468 Methionine Sulphoxide-2 0.447 Methionine Sulphoxide-1 5 1 0.468 Taurine Dimethyllysine 0.406 0.446 3-Methylhistidine 1-Methylhistidine 1 1 5 Trimethyllysine 0.386 0.386 Ammonia 0.353 3 Histidine 0.353 Glutamine 1 2 0.259 0.244 Carboxymethylcysteine 3 Glutamic Acid 2 0.228 Asparagine 1 0.206 Serine 4 pH=5.05 Galactosamine-2 3 Acid Aspartic 3 Phosphothreonine RetentionCoefficient Glucosamine-2 2 Aminoquinoline 2 γ Phosphoserine Cysteic Acid Glucosamine-1 Galactosamine-1 β Sample Type Code Compound Nameand Table 1 Aiouyi cd60.701 6 -Aminobutyric Acid 0.274 3 -Carboxy GlutamicAcid HdoysatcAi 0.186 3 Acid -Hydroxyaspartic Aiouyi cd80.734 8 -Aminobutyric Acid SOLUTIONS rpohn—1.028 0.948 1.000 0.955 0.933 0.907 0.907 — 1 acid canbefound. 1 8 0.801 Sample typecodesindicatethe ofmatrixinwhichtheamino 1 0.850 0.821 0.793 0.835 with eluentpH5.80. 1 0.779 This tablecontainstheRC’s forthederivativesusingamethodrun 6 0.689 Tryptophan Phenylalanine 1 4 1 Norleucine 1 4 0.625 Lysine 1 0.613 Leucine Isoleucine 1 0.596 Ornithine 0.640 0.589 -2 Hydroxylysine-1 1 Methionine 0.556 1 0.536 Valine 0.589 0.570 Cystine 1 0.511 0.478 Tyrosine 3 5 α Proline γ 0.426 1 0.453 1 Methionine Sulphone 0.439 3 3 0.439 Alanine 5 1 Arginine Threonine 0.363 Cysteine, Thioglycolic Taurine 1 1 0.336 5 Phosphotyrosine 2 0.350 0.324 Ammonia 0.325 0.257 Histidine 0.205 Glutamine 2 0.205 Glycine Aminoquinoline 0.194 4 Galactosamine-2 pH=5.80 3 1 0.183 3 Asparagine 1 Serine 3 Glucosamine-2 3 RetentionCoefficient Carboxymethylcysteine Hydroxyproline 2 Phosphothreonine 2 Glutamic Acid Acid Aspartic Phosphoserine Cysteic Acid Glucosamine-1 Galactosamine-1 Sample Type Code Compound Nameand Table 2 the RT ofphenylalanine. tables arethequotientofpeak’s RT dividedby (RC)listedinthe acids. Theretentioncoefficients the optimizationofseparationdesired amino The choiceofreaction conditionsisdeterminedby compounds underAccQ•Tag separationconditions. approximate retentiontimes(RT) ofdiverseamine hydrolysates. Thefollowingtablesillustratethe of aminoacidsnotpresent instandard protein in anAccQ•Tag separationforawidevariety describes thechromatographic retentionproperties unknown aminoacidelutes?Thisapplicationnote what constitutestheseadditionalpeaks,orwhere an mally foundinproteins. Howdoesonedetermine analyses, theaminecompoundofinterest isnotnor- Aiouyi cd60.651 6 -Aminobutyric Acid Aiouyi cd80.724 8 -Aminobutyric Acid Table 1 and 2 represent data from in-house and The net effect of these changes is that the pH 5.80 customer experiments. These tables may not include gradient, while slower (60 vs 45 min.), resolves data on all separations possible with Asn, Gln, Hypro and other non-hydrolysate the AccQ•Tag method. derivatives. In addition, the Ser/Glu, Arg/Thr and Cys/Tyr pairs reverse their order, the AMQ reagent Sample Codes peak moves from first to sixth in elution order, and Sample ...... Code Lys moves from before to after the Ile/Leu pair. Hydrolysate Amino Acids ...... 1 Two derivatives will likely be resolved completely ...... 2 if the difference between their retention coefficients Derivatized/Modified ...... 3 is greater than 0.015. All reported values should Samples ...... 4 be considered approximations, which may not be Non-hydrolyzed Biologic Samples ...... 5 identical for all system configurations. Physiologic Samples ...... 6 Waters AccQ•Tag Method has been shown to Novel Biochemical Intermediates ...... 7 provide successful separations of a wide variety Common Internal Standard AA’s ...... 8 of amino acids in diverse sample matrices. Separation Protocols The two separation protocols described earlier by their pH values differ in a number of other key parameters including ionic strength and triethylamine content of eluents, column temperature, and gradient profile.

220.00 Arg

210.00

200.00

190.00

His

180.00 Thr Aaba

170.00 Val Ala

160.00 Met

150.00 NH3

140.00 Tyr

130.00

mV

120.00

Gln Ile

Gly

110.00 Ser Leu Phe Pro 100.00

90.00 Asn

Asp

Glu

80.00

Orn Lys

70.00 AMQ HyPro Cys

60.00

50.00

20.00 30.00 40.00 50.00 Minutes

Further Reading Sensitive Analysis of Cystine/Cysteine using 6-Aminoquinolyl-H- Waters Corporation Hydroxysuccinimidyl Carbamate (AQC) Derivatives, 34 Maple Street Techniques in Protein Chemistry IV, 1993, Academic Press. Milford, MA 01757 (508) 478-2000 Waters AccQ•Tag Amino Acid Analysis System Operater’s FAX (508) 872-1990 Manual Number 154-02TP, Waters Corporation. http://www.waters.com/

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© 1996 Waters Corporation July 1996 Printed in the USA. WN054 DW-MP