The San Francisco Bay Area Mass Spectrometry (BAMS)
discussion
group
Thursday, October 13, 2005
6:00 pm to 9:00 pm
Michaels at Shoreline
Mountain View, CA
Dinner reservations required
by noon on Monday October 10, 2005
Unraveling the
Mystery of Spider Silk with Mass Spectrometry
Xiaoyi Hu, Ph.D.
Guidant, Corp.
Abstract
Spiders
produce multiple types of silk that exhibit remarkable mechanical
properties. In order to understand the
physical behavior of spider silk, it is essential to first elucidate
the
primary sequences of the spider silk proteins that make up the silk
fiber. In this research, we employed mass
spectrometry and reverse genetics to decode egg case silk, a silk fiber
whose
sequence was still unknown.
Egg case samples were acquired from the
black widow spider, Latrodectus hesperus. A differential
solubility assay using 8 M
guanidine hydrochloride revealed that two protein species, dubbed ECP-1
and
ECP-2, existed in the core egg case silk fiber.
Peptides generated by in-gel tryptic digestion of the two protein
species were sequenced de novo by high energy CAD MS/MS using a
MALDI/TOF/TOF mass spectrometer. The
peptide sequences obtained were confirmed by low energy CAD using a
linear ion
trap mass spectrometer. Several peptide
sequences were then used to design oligonucleotide primers, which were
used in various polymerase
chain reaction (PCR) assays. Full length
cDNA sequences of ECP-1 and ECP-2 discovered by PCR (Anchored, RACE)
were then
translated into the primary sequences of ECP-1 and ECP-2
proteins. 2D gel analysis followed by peptide mapping
using mass spectrometry further confirmed the identities of these two
protein species.
However, the amino acid composition analysis of ECP-1 and ECP-2 did not
agree well with the total amino acid composition of egg case fiber,
implying
the existence of one or more other protein species. Using a novel
sample preparation method
involving hexafluoro-2-propanol, we obtained some new peptides whose
sequences were
obtained by MS/MS analysis. PCR and cDNA
library screening were carried out based on these peptide sequences,
and a
novel fibroin, TuSp-1, was successfully identified. TuSp-1 is
composed of highly homogeneous repeats that are 184 amino acids
in length. Its amino acid composition matches closely the total
amino
acid composition of egg case fiber, indicating that TuSp-1 is the major
component of egg case silk. Two cysteine
residues were found in the N-terminal region of TuSp-1 while sixteen
cysteine
residues were located near the N-termini of ECP-1 and ECP-2.
These findings suggest that ECP-1 and ECP-2 play the role of a
cross-linking center, and that a complicated network formed by ECP-1,
ECP-2 and
TuSp-1 contributes to the silk’s mechanical properties.
Fibroin sequences of other silk will also be presented.
Background
As a
senior chemist in Guidant Corporation, Xiaoyi Hu is now working on
development
of drug-formulated medical device targeting human cardiovascular
disease. Xiaoyi obtained his Ph.D. in bioanalytical
chemistry in 2004 from University of the Pacific under the direction of
Professor Patrick Jones and David Sparkman.
His main research was focused on investigation of spider silk by mass
spectrometry. During his study in
University of the Pacific, he supported research activities in
Chemistry,
Biology Department and School of Pharmacy using mass
spectrometry. Xiaoyi also holds two
master’s degrees in Physical Chemistry, Pharmaceutical Sciences and a
B.S. from
Tsinghua University
in Beijing.
Meeting details
| Date: |
Tuesday |
Thursday,
October 13, 2005 |
| Time: |
6:00 pm |
Social hour, registration (no-host cocktails) |
|
7:00 pm |
Dinner |
|
8:15 pm |
Lecture |
| Dinner: |
featuring: |
Pork
Loin Roast, Peppercorn Sauce |
|
|
Breast of Chicken,
Florentine |
|
|
Stuffed Prawns,
Lobster Sauce |
|
|
Spinach and Cheese
Tortellini, Alfredo |
|
includes: |
Side dishes, dessert, Coffee/Tea
|
| Cost: |
$30
|
BAMS members. |
|
$40
|
Non-members. |
|
$60
|
BAMS membership plus dinner cost |
|
$15
|
Students only. |
|
$300
|
2005 Corporate Sponsors |
|
|
Dinner reservations
required
by noon on Monday October 10, 2005 |
*Note: 2005 dues need to be paid to obtain member price.
Dues ($30.00) may be paid while registering for dinner. If you
are
unsure of your 2005 Membership status, please email David Kage.
Maps & directions
Michaels at Shoreline
2960 Shoreline Blvd.
Mountain View, CA 94043
(650) 962-1014
maps.google.com
directions
Did you see or hear about the BAMS talk by Professor Murayama
entitled
"What is Mass?" What is it we are measuring after
all?
Check it out! Go to ctp.berkeley.edu
and click on "Popular Talks & Articles" for a QuickTime file.
As a service to our members, we post Employment
postings.
As a trial service to our members, we are now posting Situations
Wanted. (Note: BAMS isn't a career center and will only post
brief
positions wanted for BAMS members only).
The San Francisco Bay Area Mass Spectrometry discussion group was
formed
in 1980 to provide a regular gathering for people interested in mass
spectrometry
and allied topics. BAMS currently has a membership of about 280
individual
and 20 corporate members, and meets 8-10 times per year for a midweek
dinner
and lecture. Meetings attract between 30 and 90 people, and are
held
at a restaurant or hotel in the bay area convenient for our
speaker.
We usually convene at 6:00 pm for cocktails, dinner at 7:00 pm, and
lecture
at 8:15 pm.
If you have suggestions for a meeting topic, or know of a prominent
mass spectrometrist who will be visiting the bay area, please contact
our
program
chair.
To become a member, please fill out and mail in a membership
form.
BAMS has been fortunate to have had many excellent
speakers in our short history.
If you have suggestions for a meeting topic please contact our program
chair.
If you have questions or comments about BAMS, please contact one of
the BAMS officers.
Please thank our corporate sponsors who
help keep BAMS membership and dinner costs low.
Here are some internet links that may be of
interest to BAMS members.
If you have comments or suggestions for this web site, please email
me at hugh-gregg@llnl.gov
Last update: 9/22/2005