FR AB - Science Reference

ABRF 2001 ABSTRACTS
P81-T
How to obtain reproducible gradient capillary/nano LC for ultrahigh
sensitivity MS detection of biological macromolecus with
and without flow splitting.
Y-H. Jou, C. Wu, C. Wu, Y.W. Hong, F.J. Yang; Micro-Tech Scientific,
Sunnyvale, CA, 140 South Wolfe Road, Sunnyvale, CA 94086
The applications of gradient capillary column LC for biological macromolecular
analysis and drug discovery research have increased significantly in
recent years. As a result of this increasing need for sub-picomole detection,
capillary column LC with eletro-spray and nano-spray MS is increasingly
important.
Flow stream splitting technique introduced in 1983 by van der Wal and Yang
(1) has been utilized by many users for rapid evaluation and realization of
the advantages of capillary LC and capillary LC-MS. However, the flow stream
splitting technique has suffered from inherent poor retention time and gradient
slope reproducibility when the following condition(s) occurred: high
pressure, high split ratios, large sample amount of viscose sample solvent is
injected, column inlet flow restriction change, or splitter flow restriction
change.
Splitless flow capillary LC allows the same performance and ease of validation
for routine work as the conventional 4.6 mm id. gradient HPLC. However,
it requires each pump to deliver accurate/reproducible flow rates at
sub-�l/min. It also requires high pressure mixer(s) with small volume for fast
gradient generation and minimum solvent gradient delay time from the mixer
to the column inlet.
This poster will discuss design concepts of a new reciprocating pump that
delivers sub-�l/min flow rates for routine capillary/Nano LC applications.
Performance of the system in terms of long-term retention time reproducibility
for flow rates from 0.01 to 50 �l/min will be compared to a flow
stream splitting system. Practical considerations in terms of gradient regeneration,
system liquid end volume, mixer volume, mixing noise, and sample
clean up, desalt, and concentrating will also be discussed.
1. Sj. van der Wal and F. J. Yang, J. Resolut. Chromatogr. Chromatogr. Commun.
6, 216 (1983).
P83-M
An automated sequence assignment and multiple database
searching.
T. Sasagawa, Y. Matsumoto, M. Kojima, Y. Mizuno; Toray Res. Ctr.,
1111 Tebiro, Kamakura, Kanagawa 2488-8555, Japan
Mass spectrometry and Edman degradation are complementary to each other
and are indispensable techniques in proteomics in which systematic analysis
of a large number of expressed proteins is required. In order to facilitate
the interpretation of these data, automated data analysis and multiple database
search programs linked together are developed. PepMs is for de novo
sequencing based on a new algorithm. The sequence information can be
obtained even if a few gaps are present. Post-translationally modified amino
acid and ambiguous Edman sequence data can be used to aid the sequence
determination.
Seq is for the automated interpretation of Edman sequence data and for multiple
database searching. By correcting raw data with extraction efficiency of
PTH amino acids and with lag due to incomplete cleavage, unambiguous
sequence assignment is possible. The assigned data are automatically subjected
to database searching. A minor second sequence can be also determined.
The program also has a routine to generate theoretically possible
amino acid sequences based on observed PTH amino acids, when equal
amount of multiple sequences are observed. Using the generated pairs of
sequences, the correct pair of the sequence can be found by multiple database
searching. The result is confirmed mass spectrometry and de novo
sequence routine.
POSTER ABSTRACTS
208 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P82-S
Proteomics: identification of low abundance proteins
by MDLC/MALDI-TOF analysis.
M. Meys, S. Krishnan, K.C. Parker, M. Lin, M.D. Lynch, R. Carberry,
K. Waddell; Applied Biosystems, Foster City, CA, 500 Old Connecticut
Path, Framingham, MA 01701
The study of proteome components commonly involves the separation of a
protein extract on a 2-D gel followed by the analysis of the protein spots.
This analysis is currently being done by proteolytic digestion of the spots followed
by mass spectrometric techniques. Although this approach has proven
to be useful, it has several limitations. The main limitations are the inability
to analyze low abundance proteins, proteins of extreme pI’s and molecular
weights. We present here a method that can overcome these limitations by
eliminating the 2-D gel separation step. The process involves performing a
multidimensional chromatographic separation of the protein extract followed
by proteolytic digestion and MALDI-TOF mass spectrometric analysis of the
digests. The removal of the gel step in the process offers accessibility to all
classes of proteins, makes the process amenable for automation and enhances
speed of analysis. The use of chromatography also improves peptide
recovery. In addition, the use of the chromatographic separation enables the
removal of the highly abundant proteins from the extract and aids in the
identification of the low abundance proteins. In the present study Escherichia
coli is used as a model to validate this approach. The crude extract was
fractionated over a cation exchange column followed by further fractionation
on a reverse phase column. The fractions from the reverse phase column
were then digested with trypsin and subjected to MALDI-TOF mass spectrometric
analysis. Using this approach we identified several low abundance
proteins such as maltodextrin phosphorylase, leucine aminopeptidase, phosphate
starvation inducible protein precursor. These proteins to our knowledge
have not been located on 2-D gels of E coli indicating the validity and
usefulness of the approach.
P84-T
Amino acid specific effects on C-terminal sequencing efficiency:
comparison of activation chemistries.
D.R. Dupont1, S.W. Yuen1, R.L. Noble1, K.S. Graham2; 1Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404,
2Beckman Res. Inst., City of Hope
Efficient initial activation of the C-terminus of a protein or peptide is essential
to successful C-terminal sequencing. To facilitate the evaluation of activation
chemistries, we have synthesized sets of peptides that contain each of
the genetically coded amino acids (except proline) in three sequence positions,
at the C-terminus, penultimate to the C-terminus and third from the
C-terminus. The peptides are sequenced in groups of four or five to limit the
number of sequencing runs necessary to evaluate the behavior of each of the
amino acids in each sequence position. Included in each group are several
amino acids with reactive side chains together with amino acids with unreactive
side chains. The peptides are covalently attached to modified PVDF
membrane to minimize the effect of sample loss due to washout.
In the ATH method, the C-terminus is reacted with acetic anhydride to form
an oxazolone, which is then reacted with tetrabutylammonium thiocyanate
in the presence of TFA vapor to form a C-terminal thiohydantoin. An alternative
approach is the use of a reagent that can form thiohydantoin without
first forming oxazolone, which should minimize the potential for side reactions
and improve the sequencing initial yield. In the C-terminal sequencing
chemistry developed at the Beckman Research Institute, one such reagent,
diphenylphosphoroisothiocyanatidate (DPP-ITC), is used for activation and
thiohydantoin formation. Several years ago we presented a preliminary comparison
of these activation chemistries using several model proteins. Here we
compare the activation chemistries for all the amino acids (except proline)
using the peptide sets and outline the effects of incorporating DPP-ITC activation
into the ATH chemistry. In addition, we compare the efficiency of the
activation chemistries on a variety of proteins at different sample amounts.