AccQ•Tag interfering withtheinterpretation ofresults.Insome interfering peaks tothestandard chromatogram,potentially modified proteinsamplesoftenproduce additional In aminoacidanalysis,constituentsofnon-protein 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 Norleucine 1 Methyllysine 1 Leucine 0.695 1 0.757 Isoleucine 0.708 Lysine 3 0.737 Ornithine Norvaline 0.693 Methionine 0.656 0.664 Cysteine, Thiobutyric 1 3 Valine 7 Tyrosine 3 Cystine 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 Proline 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 Arginine 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 Glycine 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 Hydroxyproline 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 Hydroxylysine-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 amino acid 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 Glycoproteins ...... 2 if the difference between their retention coefficients Derivatized/Modified Proteins ...... 3 is greater than 0.015. All reported values should Collagen 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/
Waters and AccQ•Tag are trademarks of Waters Corporation.
© 1996 Waters Corporation July 1996 Printed in the USA. WN054 DW-MP