Phosphorylated Compounds by HPLC

Transcription

1 Phosphorylated Compounds by HPLC Featuring ERLIC, HILIC and other Ion-Exchange Techniques Table of Contents Phospholipids by HILIC Page 2 Crude Phosphorothioate by Anion Exchange 2 Phosphorylation Variants of Histone H1.5 3 Nucleotides, via ERLIC 3 Crude Synthetic Phosphopeptide 4 Isolation of Phosphopeptides via ERLIC 5 Tryptic Monophosphopeptides via ERLIC 6 Fraction of Tryptic Digest of HELA Cell Lysate via ERLIC 7 Price lists 8 1

2 Phospholipids by HILIC Column PolyWAX LP, Anion exchange, catalog number 204WX0503, 4.6x200mm, 5um, 300A Buffer A, 95% ACN, 5mM Ammonium formate (straight from Bottle, Filtered) Buffer B; 50% ACN, with 5 mm Ammonium Formate ph does not need to be adjusted, but will be around ph 6.5 and the buffers can be pre-mixed then run the gradient from 100%A to 100% B Detector ELSD at 115 C Crude Phosphorothioate by Anion Exchange Column 104WX0315, Anion Exchange, 4.6x100mm, 3um 1500A,. Buffer A: 25mM Tris, ph 8, with 30% ACN Buffer B; 1 M NaCl04 in A buffer Sample 1.7ug/25ul, flow rate 0.5ml/min Ambient temp. 2

3 Phosphorylation Variants, of HISTONE H1.5 Column; PolyCAT A,cation exchange, in HILIC Mode, catalog number 204CT0510 Highly phosphorylated forms, critical to the cell cycle, can only be detected and quantitated after separation. Phosphorylation proved to proceed in an obligate sequence of residues. -courtesy of Herbert Lindner, (Univ of Innsbruck) Nucleotides via ERLIC ERLIC of nucleic acids and nucleotides is performed with a cation-exchange column, at a ph low enough that there s no trace of a second negative charge on the phosphate groups. Otherwise, electrostatic repulsion would be so strong that none of these compounds would be retained at any level of ACN. The electrostatic repulsion most antagonizes the retention of the triphosphonucleotides, which normally elute much later than the others in either anion-exchange or HILIC. Column: PolySULFOETH- YL A (Item 204SE0503) Mobile Phase: 80mM TEAP, ph 3.0, with 84% ACN; 2ml/min 3

4 Crude Synthetic Phosphopeptide: Numerous failure sequences may not be separated by reversed-phase, but the chromatogram below demonstrates that PolyCAT A, when using an acetic acid gradient, will yield a purified target peptide; with remarkable selectivity, superior to Reversed Phase. Baseline resolution of Thr-deletion as detected by ES-MS. Column: PolyCAT A 204CT0503 Gradient 0-25% B in 60', A)10mM NH4OAc, ph 5.5 B) 40% HOAC, Flowrate 1ml/min Detection 280 nm The Sample was the main fraction collected from Reversed Phase step. The product was purified satisfactorily at the cation exchange step. Selectivity is remarkable; deletion of a Thr- residue sufficed to afford nearly baseline resolution from the main product. The elution order was as expected in some cases; Loss of -PO4 or Ser(PO4) would decrease electrostatic repulsion and increase retention time, while loss of two Arg- residues decreased retention. However, the loss of all three Arg- Residues had little effect on retention, while loss of a single Arg- actually increased retention. The indicated deletions were identified by ES-MS. Again, one cannot specify which particular residue is missing if there is more than one possiblity. 4

5 Isolation of Phosphopeptides via ERLIC* in Phosphoroproteomics (Electrostatic Repulsion-Hydrophilic Interaction Chromatography) ERLIC is a new, general-purpose mode of chromatography,; a column of the same electrostatic charge as the solutes is run in the HILIC mode. With ERLIC, phosphopeptides can be isolated selectively from tryptic digests. At ph 2.0, phosphate groups in peptides retain some of their negative charge. This does not permit isolation by Anion-exchange chromatography of singly phosphorylated peptides from tryptic digests, since the electrostatic attraction is not sufficient to overcome the electrostatic repulsion from the N- Terminus and the C-terminal Lys or Arg- Residue. If an Anion Exchange column is run in the ERLIC mode, though, then the combination of electrostatic attraction and hydrophilic interaction does suffice to pull singly phosphorylated peptides away from the nonphosphorylated peptides.peptides with multiple phosphate groups are retained so strongly that a salt gradient is necessary for elution. Example above : Separation with same column in the ERLIC and Anion Exchange Modes Note the poor retention of the singly phosphorylated fragment in the anion-exchange mode. Column, 104WX0503, 100x4.6mm, PolyWAX LP, 300A, 5um, 5

6 Tryptic Monophosphopeptides Top Chromatogram, Increasing the salt content of the mobile phase is a standard way to elute solutes in ion-exchange, and works well here. Selectivity and peak shapes are quite good. Bottom Chromatogram, An alternative gradient of decreasing ACN, switching the mode from ERLIC to Anion Exchange, since tryptic monophosphopeptides are not well retained in the anion exchange mode. The selectivity was retained although peak shapes deteriorated to some extent. The use of an ACN gradient has three significant advantages. 1. Since salt concentration doesn't vary, the absorbance baseline is steady. This suggests the possibility of monitoring 220nm for peptides that lack aromatic residues 2.Use of only 20mM ammonium formate would be quite convenient for direct flow into a mass spectrometer (a salt gradient would be necessary to elute peptides with more than one phosphate). 3. Non-ideal tryptic phosphopeptides, with more than one basic residue, elute easily with this gradient but not with the gradient of increasing ammonium formate concentration. 6

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8 Ordering Information, Catalog Number and Prices PolyCAT A, Cation Exchanger Particle Size: 5um Column IDx Length 300A Pore Size 1000A Pore Size Price.15X50mm CT CT0510 $ x150mm CT CT0510 $ x50mm CT CT0510 $ x150mm CT CT0510 $ x 50mm 051CT CT0510 $ x 150mm 151CT CT0510 $ x 100mm 102CT CT0510 $ x 200mm 202CT CT0510 $ x 35mm 3.54CT CT0510 $ x 100mm 104CT CT0510 $ x 200mm 204CT CT0510 $ x 200mm 209CT CT0510 $ x250mm 259CT CT0510 $ Javelin Guard Columns 2.1x10mm J22GCCT0503 J22GCCT0510 $80.00 each 4.6x10mm JGCCT0503 JGCCT0510 $80.00each 10x10mm Cartridge G10-CT1203 G10-CT1210 $ each, requires holder C-1000, $ Bulk Material BMCT0503 BMCT0510 $60.00/gm BMCT1203 BMCT1215 $1200/gm Syringe Cartridge UltraMicro Spin MacroSpin Column SPECT1203 $60.00 pk/10 UMSCCT1210 $60.00 pk/10 MASCCT1210 $60.00 pk/10 Lab in a Tip, Pipette Tip style TT10CAT (1-10ul) $165.00pk/96 TT200CAT, (10-200ul) pk /96 PolyWAX LP, ANION Exchanger Particle Size: 5um Column IDx Length 300A Pore Size 1000A Pore Size Price.15X50mm WX WX0510 $ x150mm WX WX0510 $ x50mm WX WX0510 $ x150mm WX WX0510 $ x 50mm 051WX WX0510 $ x 150mm 151WX WX0510 $ x 100mm 102WX WX0510 $ x 200mm 202WX WX0510 $ x 35mm 3.54WX WX0510 $ x 100mm 104WX WX0510 $ x 200mm 204WX WX0510 $ x 200mm 209WX WX0510 $ x250mm 259WX WX0510 $ Javelin Guard Columns 2.1x10mm J22GCWX0503 J22GCWX0510 $80.00 each 4.6x10mm JGCWX0503 JGCWX0510 $80.00each 10x10mm Cartridge G10-WX1203 G10-WX1210 $ each, requires holder C-1000, $ Bulk Material BMWX0503 BMWX0510 $60.00/gm BMWX1203 BMWX1215 $12.00/gm Syringe Cartridge UltraMicro Spin MacroSpin Column SPEWX1203 $60.00pk/10 UMSCWX1210 $60.00pk/10 MASCWX1210 $60.00pk/10 Lab in a Tip, Pipette Tip style TT10CAT (1-10ul) $165.00pk/96 TT200CAT, (10-200ul) pk /96 8