Your comments about the 494s being more sensitive and having better IYs
than HPs struck a nerve. I hear this comment a lot and I think the
implications are bogus. If you (meaning everyone) look at the big picture,
the HP can perform equal to or better than the 494 and here's why:
1) By the time you get in 10-15 residues, the higher HP RY compensates for
any difference in IY. Residues beyond that are just icing on the cake.
2) Proper maintenance of the HP in combination with continuous running (or
"slow running/idling") is essential to keeping the HP at peak performance.
An HP that has not been used for a week takes at least 3 weeks of continuous
running to get it back on top of its game. Been there, done that. Our's
never stop.
3) The biggest problem with the HP has been its detector system, which is
where the boost in sensitivity for the "241" came from, not from any changes
in the sequencer proper. Of course, if you installed the C-terminal
reagents, most of the benefits went away. We installed HP 1100 VWD detectors
plus semi-micro flow cells on both G1005A systems for about $6.5K per system
and saw an immediate boost in sensitivity. We now do routine peptide
identification analyses starting with 200 fmol IYs and still get the high RYs
at that level. The higher RY even allows the HP to give a cLC a run for its
money at this level for a lot less operating expense.
4) The HP Edman chemistry and HPLC methods are surprisingly robust and rarely
fail to produce usable data, even from nearly garbage samples.
The average 494 user who only runs occasionally probably does get better
overall performance than the average HP user who only runs occasionally, but
a properly maintained and continually run HP using an HP 1100 VWD detector
(not an HP 1100 DAD) can outperform a properly maintained and continually run
494. Of course, it helps if the reagents work properly.
All of this may be moot, however, since HP appears to have stopped
actively marketing its sequencer some time ago.
I get so tired of hearing HP users say "I can't get under 2 pmol". If
you can't get under 2 pmol, then you are doing something wrong, plain and
simple. Maybe it's just easier to complain about performance than to do
something about it. Based on the limited HP-user responses to my questions,
one gets the idea that most HP users don't seem to care about performance
issues. The crop you harvest has a lot to do with the seeds you planted.
Steve
====================
Stephen Tindall
Argo BioAnalytica, Inc.
====================
Subj: Re: HP Seq: Lys Recovery Problem (PDF Figs. Attached)
Date: 4/28/99 1:24:16 AM Eastern Daylight Time
From: jmackint@proteome.org.au (jim mackintosh)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
Reply-to: jmackint@proteome.org.au
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Steven,
Just some quick thoughts slightly off-topic. For our in-house work I have
found Mallingkrodt HPLC grade acetonitrile (HPs L1) and methanol (HPs L2)
work fine in the G1005As, no problems. At around AUS $60 for 4 litres it's a
huge saving. Also for our in-house work I have found that used PVDF sample
columns can be re-used with no problems. You simply re-prep them using the
PVDF 3.1 prep program and all pre-existing protein is removed. I'm yet to
attempt re-using the 3.1 protein columns but I expect this can also be done.
I'll be interested to hear how you go with your specific
problem. It may be the R2.
As I have said before I have a tremendous amount of respect for the HP
sequencers. My analogy is the HPs are like Harley Davidson's whereas the
ABIs are like Hondas. The 494s are more sensitive (they have an excellent
initial yield) and are quicker, but if you want a simple machine to mosy on
down the highway in comfort take the Harley.
Jim Mackintosh
Australian Proteome Analysis Facility, Level 4, Building F7B, Macquarie
University, Sydney, NSW, 2109, Australia
Tel: + 612 9850 6364 Fax: + 612 9850 6200 E-mail: jmackint@proteome.org.au
http://www.proteome.org.au/