Peptide Mapping

Achieve High-Sequence Coverage with SeQuant® ZIC®-HILIC

SeQuant® ZIC® HPLC columns are suitable for peptide mapping, especially so when exploring information from hydrophilic peptide fragments. Thanks to their advantageous combination of reversed phase chromatography and HILIC technology, the columns provide complementary information and may be coupled to an online 2-D peptide mapping approach.

Various Methods for Peptide Mapping

Liquid chromatography – mass spectrometry (LC-MS) is frequently used for peptide mapping, since digested cell lysate is often too complex for direct mass spectrometric (MS) analysis. Ideally, all peptides of a digested protein or protein mixture should be identified by their MS/MS spectrum and retention time. Various analytical methods have been developed, with reversed phase (RP) chromatography being the most common approach. However, the resolving power of a single RP column is not sufficient to separate and identify all peptides present.

Advantages of SeQuant® ZIC® Separation

Based on the combination of reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography, complementary HPLC columns allow much higher sequence coverage compared to traditional, one-dimensional RPLC approaches. Several studies have shown that this method provides the most orthogonal combination of separation techniques, and the highest coverage of 2-D separation space.

MilliporeSigma's zwitterionic SeQuant® ZIC® stationary phases offer distinct advantages for peptide mapping. Compared to strong cation exchange chromatography (SCX), SeQuant® ZIC® provides significantly better chromatographic resolution and prevents clustering of charged peptides. Since separation with SeQuant® ZIC® is dependent on buffer pH, analysis at low pH allows best orthogonality with RP, and resembles conventional SCX separation. However, neutral or slightly basic pH conditions enable better resolution, resulting in more comprehensive data acquisition.

HILIC eluents typically contain a high percentage of organic solvent hydrophobic peptides. Hence, they are potentially less prone to precipitation during sample handling on the column than in RP chromatography. This means that HILIC columns, despite being mostly applied for analysis of hydrophilic molecules, can also be used to identify hydrophobic peptides not detected with a RP column. Another feature of HILIC is that higher sensitivity can be expected for many peptide sequences. This is also due to the high percentage organic content in the mobile phase, which enhances volatilization in the detector.

The use of SeQuant® ZIC® technology for peptide mapping has been shown with three trypsin digested proteins: Bovine Serum Albumin (BSA), Cytochrome C, and Ovalbumin. The results demonstrate higher sequence coverage, thus better characterization of the proteins. Links to documents and results can be found in the "Further Reading" section below.