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Tetaz and colleagues
1
developed a method which allows chromatogra-
phing various intact, soluble proteins by HILIC. The separation of intact
proteins was demonstrated with two different lipophilic proteins, human
apoA-I, recombinant human apoM, plus a mitochondrial, membrane-
associated protein, equine cytochrome C. Five different commercially
available HILIC columns were compared. The figure below shows a
separation of intact proteins on TSKgel Amide-80. All of the columns
tested seemed to indicate that apoM could be separated into multiple
isoforms. To prove the hypothesis that these isoforms represented
different glycosylated isoforms of apoM, the TSKgel Amide-80 column
was chosen for further studies. Mass spectrometric analysis of the
fractions obtained revealed that HILIC is able to separate the different
glycosylated isoforms of apoM from each other and from the non-
glycosylated form.
A similar column was used by Solyakov et al.
2
to fractionate tryptic
peptides when analyzing the role of protein phosphorylation in the life cycle
of malaria parasites such as Plasmodium falciparum. In order to provide a
comprehensive picture of malarial phospho-proteome, they undertook a
mass spectrometry-based global phospho-proteomic study. Soluble tryptic
peptides were fractionated on a TSKgel Amide-80 HILIC column 4.6
× 250 mm, 5 µm and used for immobilized metal affinity chromatography
(IMAC). IMAC flow through and eluted fractions were then analyzed by
LC/MS-MS. 1177 phosphorylation sites on 650 parasite proteins involved
in a wide range of general cellular activities were identified in this study.
TSKgel Amide-80 was also successfully applied for the HILIC separation
of steviol glycosides by Zimmermann et al.
3
Glycoside extracts of the
species Stevia rebaudiana, commonly known as sweetleaf, sugarleaf,
or simply stevia gathered attention with the increasing demand for low-
carbohydrate, low-sugar food alternatives. Since 2011 purified stevia
extracts are allowed to be marketed as sweeteners in Europe. Stevia
rebaudiana Bertoni contains several steviol glycosides with sweet flavour,
which are all sweeter than sucrose (up to factor 450). The various steviol
glycosides are difficult to separate by reversed phase chromatography.
In this paper, five different hydrophilic liquid interaction chromatography
columns were characterized using isocratic elution (5–20% water in
acetonitrile with buffer or formic acid). Separation of the steviol glycosides
was possible with all but one of the tested columns, but the robustness of
the separation against changes of buffer concentration and percentage of
water differ. Aqueous percentage and ion strength of the eluent are themain
factors to be optimized in method development. The TSKgel Amide-80
2 x 150 mm, 3 µm column showed the highest robustness against
changes in buffer concentration and water content of the mobile phase.
HILIC Applications for Proteins, Phosphopeptides
and Steviol Glycosides
Hydrophilic interaction liquid chromatography (HILIC) is the preferred method for the separation of polar
compounds. Analysis of glycans, carbohydrates, peptides, polar drugs and THEIR metabolites, vitamins and other
hydrophilic compounds are typical HILIC applications. For years TSKgel Amide-80 HPLC columns have been used
successfully for HILIC separations of polar compounds, documented in more than 250 scientific publications.
We cite here some recent publications where TSKgel Amide-80 columns were applied for the separation of intact
proteins, tryptic phosphopeptides and steviol glycosides, respectively.
06
TSKgel
IN THE LITERATURE
HILIC SEPARATION OF HUMAN APO M (BLACK), HUMAN APO A-I (RED) AND
EQUINE CYTOCHROME C (BLUE) ON A TSKgel AMIDE-80 COLUMN
Column: TSKgel Amide-80 (4.6 x 250 mm, 5 µm); Flow rate 1 mL/min; Elution:
A: 50 mM ammonium formate (pH 3.7)/hexafluoroisopropanol/isopropanol/
acetonitrile (24.5/0.5/50/25), B: 50 mM formic acid (unbuffered = pH 2.5)/hexa-
fluoroisopropanol/isopropanol (89.5/0.5/10) + 4–5 mg/L l-tryptophan; Gradient:
0 - 10 min: 0% B,10 - 40 min: linear gradient 0 - 100% B, 40 – 50 min:100%
B; Temperature: 24°C; Detection: UV@215/254/280 nm; Samples: purified proteins
(100 µg diluted in A)
References:
[1]
Tetaz, T.; Detzner, S.; Friedlein, A.; Molitor B. & Mary J.-L. Hydrophilic Interaction
Chromatography of Intact, Soluble Proteins. J. Chrom. A. 1218 (35) 2011: 5892-5896
[2]
Solyakov, L. et al. Global Kinomic and Phospho-proteomic Analyses of the
Human Malaria Parasite Plasmodium falciparum. Nat. Commun. 2:565 doi: 10.1038/
ncomms1558 (2011).
[3]
Zimmermann, B.F.; Woelwer-Rieck, U. & Papagiannopoulos, M. Separation of
Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography.Food Anal.
Methods 2011