Paul Jeno (Basel) has packed capillaries of PolyHYDROXYETHYL Aspartamide and
used them for on-line ESI-MS. It worked, but only if one loaded peptides on
the capillaries in a higher level of acetonitrile (ACN) than would be
necessary to get decent retention on a regular HPLC column (@ 90-95% vs. 80%)
(NOTE: In HILIC, the higher the initial ACN level, the better the retention].
I suspect this reflects a tendency of LC-MS labs to overload capillaries.
Ion-exchange materials are more forgiving of this, but HILIC materials are
not.
Kristine Swiderek et al. have also used capillaries of PolyHYDROXYETHYL A
(Tech. in Protein Chem. 6 (1995) 267) as a method of removing detergents from
samples on-line. They too complained about inadequate retention of several of
their peptides. The problem is probably their use of 0.1% TFA in 90% ACN for
the starting mobile phase. TFA promotes retention in RPC but has the opposite
effect in HILIC. A better electrolyte to use is 10 mM ammonium formate, pH
2.7-3. This gives a lumpy baseline in UV at 220 nm, but is fine for ESI-MS.
Steve Carr has used microbore PolyHYDROXYETHYL A columns for LC-ESI-MS; he's
presented results at conventions but hasn't published anything yet. He uses a
gradient from 85 - 0 % ACN in 10 mM ammonium formate. He made the intriguing
observation that one can resolve glycopeptides from nonglycopeptides
selectively in a tryptic digest this way (he monitors the Hex-HexNAc pair and
sialic acid to ascertain the location of the glycopeptides).
Several papers present applications of HILIC with subsequent off-line mass
spec; these could just as easily have been performed on-line:
1) Zhang and Wang, J. Chromatogr. B, 712 (1998) 73. Tryptic digestion of
recombinant interferon with subsequent RPC mapping. The two glycopeptide
peaks were collected and rerun by HILIC, which resolved the glycosylation
variants. These were characterized by MALDI-TOF.
2) Lane et al., J. Cell Biol. 125 (1994) 929. A tryptic digest of SPARC
protein was resolved by HILIC; the presence of minute amounts of GHK and KGHK
was demonstrated by ESI-MS of fractions collected from the elution window of
synthetic standards of these peptides.
3) Jeno et al., Anal. Biochem. 215 (1993) 292. HILIC of an electroeluted
integral membrane protein to get rid of SDS (NOTE: Used 50 mM formic acid in
the mobile phase instead of ammonium formate).
The take-home message from all this: You can use HILIC-ESI-MS but do use
formate or acetate salts (or just dilute formic acid) in the mobile phase
instead of TFA, use a lot of ACN when loading your capillaries or microbore
columns, and don't overload them.
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: 410-992-5400 FAX: 410-730-8340 e-mail: PolyLC@aol.com