1) Many gradient separations become isocratic separations;
2) Phosphopeptides can be isolated selectively from tryptic digests and separated with high resolution.

Ionizable biochemicals with a high charge are generally significantly better retained in chromatography than compounds of low charge (e.g. ATP vs. AMP), and those charges tend to dominate the mode of interaction with the surface chemistry to the detriment of other functional groups. This is true in both ion-exchange and hydrophilic interaction chromatography (HILIC). A gradient is necessary to make both types of compounds elute in the same time frame. However, if HILIC is performed using an ion-exchange column of the same charge as the most highly-charged functional groups, then their retention is selectively antagonized by electrostatic repulsion (ERLIC). This permits their elution in the same time frame as less highly-charged compounds using isocratic conditions, or with lower amounts of buffer, and it allows the chromatography to enhance the presence of oppositely charged or neutral polar functional groups within the molecule.

The polarity of the surface chemistry of the PolyHYDROXYETHYL A™ column ( poly-2-hydroxyethyl aspartamide) is modifiable by pH. In this respect it is analogous to a weak ion exchange chemistry, where elution can be effected by either a pH change or by solvent polarity (buffer strength or organic solvent content). By selecting a pH of 2.7, 4.4 or 6.5 one can select a surface polarity which can compliment the polarity of the functional group in one's molecule to enhance selectivity.

In contradistinction to the PolyHYDROXYETHYL A, the zwitterionic, ZIC®-HILIC, and the inverted zwitterion, ZIC®-cHILIC, stationary phases maintain their charge over a wider pH range, analogous to a strong ion exchange chemistry. This provides a stable surface chemistry against which the polarity of the analytes alone can be modified, when trying to affect selectivity by operating at different pH's.

Schematic illustation of the zwitterionic, ZIC-HILIC, betain sulfonate stationary phase.

Schematic illustation of the inverted zwitterionic, ZIC-cHILIC, cholinate stationary phase.

A. Isocratic and Gradient Separations:

Amino acids, peptides and proteins: The most polar groups in this family of molecules are the basic, amino residues. If ERLIC is performed using a column with an amine functional group, an anion-exchange column (e.g., PolyWAX LP™) at a pH ~ 2.0 (low enough for carboxyl groups to lose their negative charge), then amino acids, peptides and proteins will have a net positive charge and will thus experience some degree of electrostatic repulsion. The most basic compounds, normally the best-retained in HILIC, experience the most repulsion in ERLIC.





Nucleotides and nucleic acids: If ERLIC is performed with a negatively charged surface chemistry, a cation-exchange column (e.g. PolySULFOETHYL A™) with TEAP buffer at a pH < 3.5, a pH at which phosphate groups have just a single negative charge [note: electrostatic repulsion is too great at a higher pH where phosphate groups acquire a second negative charge], then the long eluting tri-phosphates can be eluted with significantly less buffer, even as little as 10mM. Use of triethylamine methyl phosphonate buffer salts, which aren't volatile either, significantly increases the retention of tri-phosphorylated species vs. triethylamine phosphate. While a HILIC separation of these molecules is possible using a gradient, it requires up to 250mM ammonium acetate to elute the tri-phosphates, and the phosphate groups dominate the separation order.



However, with the use of a polymer based, zwitterionic, HILIC chemistry (ZIC®-pHILIC) at a pH above the pKa of the amine of the betain sulfonated surface, a negative surface chemistry is formed. This permits a similar Adenosine Phosphate analysis (AMP, ADP, ATP, CoA, Acetyl-CoA, and Pantothenate) using gradient ERLIC LC/MS conditions, but with volatile buffers (80% ACN, 20% 10 mM Ammonium Carbonate, 0.2% NH₄OH).

B. Selective Isolation:

ERLIC for Selective Isolation of Phosphopeptides:
At pH 2.0, phosphate groups in peptides retain some of their negative charge. This does not permit the 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 toward the basic functional groups of the column. However, phosphate residues are quite hydrophilic.

In the ERLIC mode, the combination of electrostatic attraction and hydrophilic interaction does suffice to pull singly phosphorylated peptides away from the non-phosphorylated peptides in tryptic digests, at pH 2. Also, unlike the situation with high-affinity media such as IMAC or titania, the phosphopeptides are well-resolved from each other. This permits their convenient separation into numerous fractions, an important tool in phosphoproteomics for identifying the sequences of thousands of phosphopeptides from a single sample. Peptides with multiple phosphate groups are retained so strongly that a salt gradient is necessary for elution as shown in the following two examples:

ERLIC separation of phospho-peptides from HeLa cells using a PolyWAX LP column, P104WX0503. It should be noted that the recovery of phosphopeptides is highest if low-salt fractions are desalted with HyperCarb® media and high-salt fractions are desalted using RPC, C-18 media.

Subtractive Isolation of Phosphopeptides:
Use of an SCX chemistry (e.g., PolySULFOETHYL A™) for fractionation of a tryptic digest of a phosphorylated protein can segregate the phosphorylated peptides. They elute at the beginning of the gradient, while the high capacity SCX column holds up the more positively charged species. Take care to lyophillize the ammonium bicarbonate two to three times with methanol to reduce the buffer-salt content of the sample, otherwise doubly charged peptides can contaminate these single charged peptides in the first fraction.

Comparison of SCX, TiO, HILIC (HEA) and HILIC (WAX) for Phosphopeptide segregation and/or fractionation shows the value of ERLIC surfaces for reducing the amount of buffer necessary for eluting phosphopeptides compared to using other HILIC surface chemistries.

Copyright© 1995-2008 The Nest Group, Inc. All rights reserved (established 1984)

---

For more information or to place an order contact:
The Nest Group, Inc. · 45 Valley Road · Southborough, MA  01772-1323
Tel: 800-347-6378 or 508-481-6223 · Fax: 508-485-5736
For your convenience, we accept Mastercard, VISA, and American Express credit cards.

---

| Home | IdeaBook | Orders | Price/Applic | Price/Vendor | Protocols|

If you have problems or comments concerning our WWW service, please send an e-mail to Amos Heckendorf, webmaster.
  Legal | Trademarks

Last Updated: 03/17/08