Methods for producing and analyzing biopolymer ladders

Methods of producing biopolymer ladders and their use to obtain structural information about the biopolymer. The ladders are produced by setting up catalytic cleavage and terminating reactions at the end of biopolymer molecules. The terminating reactions terminate cleavage of a percentage of the biopolymer molecules at each round of cleavage.

BACKGROUND OF THE INVENTION 
The present invention relates to the field of biochemistry. In particular, 
it relates to methods of creating biopolymer ladders useful for obtaining 
structural information about the biopolymer, among other things. 
The analysis of the structure of various biopolymers is an area of great 
importance in biochemistry. Molecular genetics depends on a knowledge of 
the nucleotide sequence of DNA or RNA molecules. The amino acid sequence 
of proteins provides information useful for studying their function and 
for isolating the nucleic acids that encode them. There is an increasing 
recognition of the importance of post-translational protein modifications 
in the coordination and control of biological processes at the molecular 
level. In particular, many cellular processes, including cell division, 
are controlled by selective and reversible phosphorylation of proteins. 
More recently, as carbohydrates have proven to be of importance in various 
biological functions, their structure has become a focus of attention. 
Various artificial polymers are also being developed to mimic the function 
of the natural biopolymers. 
Various strategies exist for analyzing the structure of biopolymers. The 
most commonly used method of determining the sequence of nucleic acids, 
the dideoxy method, involves creating four sets of sub-sequences of a DNA 
molecule that terminate at each of the four bases, separating the 
fragments by polyacrylamide gel electrophoresis (PAGE), and using 
autoradiography to read out the sequence. By contrast, analysis of 
carbohydrate structure involves a laborious process of repetitively 
cleaving monosaccharides from a polysaccharide, and determining the 
identity of each monosaccharide removed. 
A commonly used method of peptide sequencing, Edman degradation, involves 
sequentially cleaving amino acids from the amino-terminus of a peptide, 
and then determining the identity of the amino acid cleaved. However, 
traditional Edman degradation gives no information concerning 
post-translational modification of polypeptides. Additionally, Edman 
degradation cannot be used for C-terminal sequencing. Such information is 
crucial for correlating gene structure and protein function. 
Peptide sequencing has also been attempted using exopeptidases to remove 
terminal amino acids from a polypeptide. Such methods involve removing 
samples of the polypeptide during the time course of digestion, 
terminating the action of the exopeptidase, and analyzing the resulting 
collection of samples. However, confusion can result from the widely 
varying rates at which the enzyme cleaves the peptide bond between 
different amino acids, which can cause gaps in the collection of peptides 
produced. 
SUMMARY OF THE INVENTION 
The present invention provides, for example, methods for producing a 
biopolymer ladder. In a first step, an ensemble of biopolymer molecules is 
contacted with a terminating reagent that reacts with the biopolymer 
molecules to add a terminating moiety, and with a cleavage reagent that 
catalyzes the removal of terminal units from the end of a biopolymer. 
According to one aspect of the invention, the rate of removing terminal 
units from biopolymer molecules bearing a terminating moiety is less than 
the rate of removing terminal units from biopolymer molecules that do not 
bear a terminating moiety. The biopolymers are contacted with the reagents 
under conditions and in amounts so as to create a collection of 
biopolymers comprising a nested set. 
According to certain embodiments of this invention, the biopolymer is a 
polypeptide, the cleavage reagent is a carboxypeptidase, the terminating 
reagent is an amino acid amide, and the rate at which amino acids are 
removed from the polypeptide is faster than the rate at which the amino 
acid amide is added to the polypeptide. In certain embodiments, the 
cleavage reagent is thermostable. 
This invention also provides biopolymer ladders comprising a nested set of 
at least two and preferably at least three biopolymer molecules wherein 
the members bear the same terminating moiety added catalytically. 
This invention also provides methods of obtaining information about the 
structure of a biopolymer involving preparing a biopolymer ladder 
according to the methods of this invention, determining the differences in 
mass between the members of the ladder, and determining the identity of 
units whose masses correlate with the determined mass differences. 
According to one embodiment of the invention, the biopolymer is a 
polypeptide, the cleavage reagent cleaves a C-terminal or N-terminal 
peptide bond of polypeptides, thereby removing a terminal amino acid, and 
the method produces information about the sequence of amino acids in the 
polypeptide. According to another embodiment of this invention, the method 
of determining mass differences involves subjecting the biopolymer ladder 
to mass spectrometry analysis.

DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a method for the generation of biopolymer ladders 
via controlled digestion of a formed polypeptide using a catalytic agent. 
Biopolymer ladders are useful for obtaining structural information about 
the parent biopolymer. In one embodiment of the invention, the biopolymer 
ladder is composed of nested fragments of a parent biopolymer (optionally 
including the parent) that differ in length by one monomer unit each. 
These ladders are useful for determining the sequence of units in the 
parent biopolymer by, for example, determining the difference in mass 
between adjacent members of the ladder. The difference corresponds to, and 
aids in the identification of, the unit removed to produce the shorter 
fragment. 
The methods of this invention involve setting up competing reactions 
directed to the terminus of the biopolymer molecules. One reaction is the 
removal of terminal units from the biopolymer molecules by a cleavage 
reagent, such as the hydrolysis of a terminal amino acid from a peptide by 
an exopeptidase. The other reaction is the reaction of the biopolymer 
molecules with a terminating reagent that results in addition of a 
terminating moiety to the biopolymer. The reactions are selected so that 
the rate for removing a terminal unit or units from biopolymers bearing a 
terminating moiety is slower than the rate for removing a terminal unit or 
units from biopolymer molecules that do not have terminating moieties. 
Thus, once a terminating moiety has been added to a biopolymer by reaction 
with the terminating reagent, the modified terminal unit is essentially 
refractory to cleavage by the cleavage reagent. 
One embodiment of this invention takes advantage of the fact that catalysts 
lower the activation energy for reactions in both directions, bringing the 
system to an equilibrium defined by the relative concentration and 
chemical potentials of the reactants. In this embodiment, the terminating 
reagent is added to the system in molar excess, so that the equilibrium of 
the reaction of adding or removing the terminating moiety to or from the 
biopolymer is significantly in the direction of adding the terminating 
moiety. When the system is properly optimized, for any particular fragment 
size a terminating moiety is added to some fraction of the biopolymers and 
a terminal unit or units are cleaved from some other fraction. Those 
biopolymer fragments that are capped with a terminating moiety tend to 
resist further cleavage. Those that are not capped will be available for 
cleavage and subsequent capping. By allowing this reaction to proceed, a 
set of truncated fragments of the starting biopolymer is formed with the 
members of the set differing in composition by the same number of monomer 
units. 
As used herein, the term "biopolymer" means a polymer made of a sequence of 
biochemical monomer units. Biopolymers include, for example, peptides, 
nucleic acids, complex carbohydrates, including analogs of these 
biopolymers, as well as other products of the sequential addition of 
biochemical monomer units. This includes, for example, peptides containing 
D- or L-amino acids, amino acid analogs and peptidomimetics that induce 
alpha helices, beta- or gamma-turns or other structural elements and 
nucleic acids containing nucleotide analogs. 
As used herein, the term "biopolymer ladder" means a nested set of at least 
two and preferably, at least three, biopolymer molecules. As used herein, 
a "nested set" means a set in which the sequence of units of each member 
in the nested set, except, of course, the largest member, is contained in 
the sequence of a larger member in the set. The difference in size between 
any members of a nested set normally equals the number of terminal units 
removed in a single cleavage reaction. When the set contains at least 
three members, and the size of each member in the set differs from the 
next largest member by the same number of terminal units. For example, a 
biopolymer ladder created from a biopolymer with the sequence 
A-B-C-D-E-F-G-H-I, could include, for example, the nested set of 
biopolymers: A-B-C-D-E-F-G-H-*, A-B-C-D-E-F-G-*, A-B-C-D-E-F-*, 
A-B-C-D-E-*, A-B-C-D-*, and A-B-C-*. ("*" indicates a terminating moiety.) 
In this set, the biopolymers differ in length by one biopolymer unit. 
Another example of a biopolymer ladder created by the action of a cleavage 
reagent that removes two terminal units at a time from the biopolymer 
could be, for example, A-B-C-D-E-F-G-*, A-B-C-D-E-* and A-B-C-*. In this 
set, the biopolymers differ in length by two units. In the case of a 
branched biopolymer, such as a complex carbohydrate, a sequence is 
contained in another sequence if the structure of the larger branched 
structure is the same as the smaller branched structure except for at 
least one terminal unit, e.g., a terminal sugar moiety. 
As used herein, the term "cleavage reagent" means a reagent containing a 
catalytic activity capable of catalyzing the removal of monomer units from 
a biopolymer. In one embodiment of the invention, the cleavage reagent is 
an enzyme. Other cleavage reagents include, for example, acids, bases, or 
naturally occurring products. The cleavage reagent can catalyze the 
removal of a single unit or multiple units from the end of the biopolymer. 
When the biopolymer is a peptide the cleavage reagent can include, for 
example, exopeptidases, i.e., carboxypeptidases or aminopeptidases. Useful 
exopeptidases include mono-peptidases and poly-peptidases, such as 
di-peptidases and tri-peptidases. This invention contemplates, in 
particular, the use of carboxypeptidase Y, carboxypeptidase P, 
carboxypeptidase A and carboxypeptidase B. It also contemplates the use of 
aminopeptidases, such as leucine aminopeptidase, microsomal peptidase, and 
so forth. 
Certain peptidases catalyze the breaking of the peptide bond between 
certain amino acids faster than the peptide bond between other amino 
acids. For example, carboxypeptidase A cleaves the peptide bond between a 
carboxylterminal proline and other amino acids (i.e., NH.sub.2 --AA.sub.1 
-- . . . --AA.sub.n --Pro--COOH) much more slowly than the peptide bond 
between other amino acids at the carboxyterminus. Therefore, in certain 
embodiments of the invention, the cleavage reagent can be a cocktail, 
including more than one peptidase, each with different specificities so 
that the rate of cleavage of peptide bonds is more uniform than the rate 
using any one of the cleavage reagents alone. One example of this is the 
use of a mixture of carboxypeptidase A and carboxypeptidase Y to produce a 
peptide ladder in which all the members of the nested set of fragments are 
represented. 
Proteins and other biopolymers can have secondary or tertiary structure 
that leaves the terminal residues unaccessible to the cleavage reagent. 
Accordingly, this invention provides means and methods for exposing the 
termini of biopolymer molecules to allow the cleavage reagent access to 
the termini, for example, by denaturing the biopolymer molecules. One 
embodiment of this invention involves contacting the ensemble of 
biopolymer molecules with a thermostable cleavage reagent. As used herein, 
the term "thermostable cleavage reagent" refers to a cleavage reagent 
having an optimal activity at a temperature above 50.degree. C. In this 
method, the ensemble of biopolymer molecules is contacted with the 
cleavage reagent at a temperature that will at least partially denature 
the biopolymer molecule, for example by disrupting secondary and/or 
tertiary structure. For many proteins, one can adequately denature the 
molecule so as to expose a terminus by exposing the molecules to 
temperatures above about 70.degree. C. Normally, reactions will be carried 
out below about 100.degree. C. However, the temperature can be chosen with 
reference to the optimal reaction temperature of the cleavage reagent. For 
example, a reaction temperature of 90.degree. C. may be preferable in the 
case of a thermostable enzyme whose optimal reaction temperature is 
90.degree. C. 
Thermostable enzymes such as those found in thermophilic bacteria are 
useful in this invention. Several thermostable carboxypeptidases have been 
identified in and isolated from thermophilic bacteria. For example, 
carboxypeptidase Taq, found in Thermus aquaticus YT-1, has an optimum 
reaction temperature of 80.degree. C. (S.-H. Lee et al. (1994) Biosci. 
Biotech. Biochem. 58:1490-1495.) Thermostable aminopeptidases have been 
identified in and isolated from Bacillus stearothermophilus (Y. Sakamoto 
(1994) Biosci. Biotech. Biochem. 58:1675-1678); Streptococcus thermophilus 
(M. P. Chapotchartier (1994) Eur. J. Biochem. 224:497-506); Streptococcus 
salivarius subsp. thermophilus (amino peptidase N) (R. G. Midwinter (1994) 
J. Appl. Bacteriol., 77:288-295); and Sulfolobus solfataricus (M. Hanner 
et al. (1990) Biochem. et Biophys. Acta 1033:148-153). 
It will be clear to anyone skilled in the art that other cleavage reagents 
having desirable properties can be isolated from organisms living in 
extreme environments and used in the methods of this invention. For 
example, salt-tolerant cleavage reagents can be isolated from halophilic 
bacteria or archeabacteria. Salt-tolerant cleavage reagents are useful for 
preparing biopolymer ladders from biopolymers, especially proteins, that 
are denatured under high ionic strength conditions. 
In the case of nucleic acids, the cleavage reagent can include, for 
example, 3' or 5' exonucleases such as Bal-31 nuclease, E. coli 
exonuclease I, E. coli exonuclease VII, E. coli DNA polymerase I 
exonuclease activity, the exonuclease activity associated with Klenow 
fragment of DNA polymerase I, an exonuclease activity associated with T4 
coliphage DNA polymerase, and so on. 
In the case of carbohydrates, the cleavage reagent can include, for 
example, exoglycosidases such as a variety of fucosidases, mannosidases, 
galactosidases and so forth. 
As used herein, the term "terminating reagent" means a reagent that, upon 
reaction with a biopolymer, adds a "terminating moiety" to the biopolymer 
that slows or prevents the reaction of the cleavage reagent with the 
biopolymer. That is, the reaction for removing a terminal unit or units 
from a biopolymer bearing a terminating moiety is slower than the reaction 
rate for removing a terminal unit or units from a biopolymer molecule that 
has not reacted with a terminating moiety. When the cleavage reagent is an 
enzyme, the maximum velocity for removing terminal units from a biopolymer 
bearing a terminating moiety is slower than the maximum velocity for 
removing a terminal unit from a biopolymer not having a terminating 
moiety. 
In one embodiment of this invention, the cleavage reagent catalyzes both 
the cleavage of terminal units and the addition of the terminating moiety, 
e.g., when the cleavage reagent is an enzyme. In this case, the 
terminating reagent can be selected to have a structure sufficiently 
different from the original units of the biopolymer that the rate constant 
for adding/removing the terminating reagent is slower that for 
adding/removing the original units. According to a preferred embodiment of 
the invention, the rate at which non-terminated units are removed from the 
biopolymer molecules is greater than the rate at which terminating 
moieties are added to terminal units of biopolymer molecules. 
For example, in the case of peptides in which the cleavage reagent includes 
a carboxypeptidase, the terminating reagent can be an amino acid amide. 
L-lysinamide has at least two desirable properties as a terminating 
reagent for use in peptide ladder generation. First, L-lysinamide is 
highly water soluble. This allows its use at high molar concentration. 
Secondly, L-lysinamide contains a basic moiety and, therefore, may enhance 
peptide ladder detection when the means to be used for analyzing the 
peptide is MALDI mass spectrometry because basic groups can aid in 
ionization. Because the amino acid amides have amide groups in place of 
carboxyl groups, carboxypeptidases catalyze their addition/removal from 
the carboxy-terminus of a peptide at rates several orders of magnitude 
less than ordinary amino acids. For example, carboxypeptidase Y catalyzes 
the hydrolysis of C-terminal peptide bonds of peptides at rates up to 
three orders of magnitude greater than for terminal amides when comparing 
the kcat/KM for the hydrolysis of FA-Phe-Leu-OH and FA-Phe-Gly-NH.sub.2 
(Raaschou-Nielsen et al. (1994) Peptide Research 7:132-135). Thus, for all 
practical purposes, once an amino acid amide is added to the 
carboxy-terminus of a peptide, the carboxypeptidase will not remove it as 
it continues to cleave terminal amino acids from other peptide molecules. 
When the cleavage reagent is an amino peptidase, the terminating agent can 
be an n-acetyl amino acid, such as n-acetyl lysine. 
In a variation of these methods, the terminating reagent can include a 
mixture of isotopes. Biopolymer ladders containing such mixtures are 
useful in detecting members of the ladder. For example, members of the 
ladder bearing termination moieties that differ in mass can be identified 
by split peaks in mass spectrometry (W. R. Gray (1970) Biochem. Biophys. 
Res. Commun. 41:1111-1119). Members of ladders bearing radioactively 
labelled terminating moieties also can be identified by autoradiography. 
Methods of producing biopolymer ladders involve optimizing the reaction 
system by using appropriate amounts of the reagents and selecting reaction 
conditions so as to form a ladder. This involves using a sufficient amount 
of terminating reagent so as to produce a detectable amount of terminated 
biopolymer at each sequential cleavage step. This amount depends, of 
course, on the sensitivity of the detection means selected. Typically, the 
terminating reagent is added in molar excess so as to significantly drive 
the reaction in the direction of adding terminating moieties. According to 
one embodiment, terminating reagent is added in an amount so that several 
percent of the biopolymers are terminated in each reaction. A range of 
useful amounts of ladder members can easily be determined empirically. 
Other factors that can be modulated to set appropriate conditions include 
temperature, pH, and buffer system. Generally speaking, when the cleavage 
reagent is an enzyme, conditions under which the enzyme would normally be 
used will provide a good starting point for further optimizing of reaction 
conditions. Additionally, in the case that a cocktail of enzymatic 
cleavage reagents is to be used, it is useful to select compatible 
enzymes. Taking pH as an example, in many cases it will be useful to 
select enzymes which have similar activity profiles as a function of pH, 
however there may be cases in which it is desirable to use an enzymatic 
cleavage reagent at less than its optimal pH in order to control substrate 
preference effects. 
The biopolymers subjected to the methods of this invention can have a 
variety of sizes. However, the impact of the average uncertainty in 
measured mass becomes more significant as the molecular mass increases. 
Below 3500 daltons, the mass deviation is less than .+-.0.3 dalton, and 
there is no ambiguity in distinguishing even the most closely related 
pairs of monomers and, in particular, amino acids (Leu/Ile have identical 
mass). However, above 3500 daltons, uncertainties of 0.4 to 0.9 dalton 
introduce certain ambiguities in the identification of monomers of closely 
similar residue masses. For polypeptides that do not contain any amino 
acids having similar mass to other amino acids, this uncertainty will 
cause no problem at all. Even if there is some ambiguity in the identity 
of certain monomers in the polymer, the method of the invention still 
provides useful sequence information. Accordingly, in certain embodiments 
of the invention, the biopolymer has mass below about 5 kDa and, 
preferably, below about 3.5 kDa. When the biopolymer is a polypeptide, the 
polypeptide can have fewer than about 50 amino acids and, preferably, 
fewer than about 35 amino acids. Valuable sequence information can be 
obtained from polypeptides having larger sizes, as well. 
This invention also provides the biopolymer ladders produced by the methods 
of this invention. These ladders are characterized by a collection of 
biopolymers having at least two, and preferably three members in a nested 
set wherein the members bear the same terminating moiety. In particular, 
this invention is directed to ladders having a catalytically added 
terminating moiety such as, in the case of peptides, an amino acid amide 
such as lysinamide. 
Biopolymer ladders can yield useful structural information about the 
biopolymer, including for example, the sequence of monomer units. When the 
cleavage reagent cleaves one unit from the biopolymer at a time, the 
members of the ladder will differ in mass by the mass of the unit removed, 
thereby providing information about the identity of the removed unit. 
Ladders created by the action of catalysts that remove more than one 
monomer unit from a biopolymer at a time also provide useful information. 
For example, digesting a protein with trypsin will leave either lysine or 
arginine at the carboxy-terminus. When the resulting peptide is digested 
with a di-peptidase in the methods of this invention, the mass of the 
first two units removed will equal the mass of arginine or lysine and the 
mass of the penultimate amino acid, thus helping to yield its identity. 
The identity of several amino acids at either end of a protein often will 
suffice to prepare primers for PCR useful in amplifying a gene encoding 
the protein. Even incomplete sequence information can supplement 
information from other sources. 
Accordingly, this invention is also directed to methods of obtaining 
sequence information about a biopolymer. The methods involve preparing a 
biopolymer ladder comprising a collection of biopolymers in a nested set 
according to the methods of this invention, determining the differences in 
mass between the members, and determining the identity of units whose 
masses correlate with the determined mass differences. The difference in 
mass between members of the ladder aids in identifying the removed units. 
According to one embodiment, this invention provides a method for 
determining the sequence of amino acids in a polypeptide. The method 
involves creating a polypeptide ladder having a nested set of peptides in 
which each of the peptides differs in length from another peptide in the 
set by one amino acid, determining the difference in mass between the 
peptides in the nested set, and determining the identity of amino acids 
whose masses correlate with the determined mass differences. The identity 
of the amino acids in the nested set indicates the sequence of amino acids 
in the peptide. This method does not require determining the amino acid 
sequence of the entire polypeptide. Partial sequences are also useful. 
This invention contemplates several methods of determining mass differences 
between members of a biopolymer ladder. According to one embodiment of 
this invention, the mass differences between members of the ladder are 
determined by mass spectrometry. Methods for mass spectrometry include, 
for example, .sup.252 Cf plasma desorption, electrospray ionization, fast 
ion bombardment, chemical induction decay and, in particular, 
matrix-assisted laser desorption mass spectrometry ("MALDI-MS"). Methods 
of performing mass spectrometry on biomolecules are well known in the art. 
Apparati and methods for performing MALDI-MS are described in International 
Publication WO 93/24835 (U.S. patent application Ser. No. 08/341,555), 
U.S. Pat. No. 5,288,644, R. Beavis and B. Chait (1990) PNAS 87:6873-6877, 
B. Chait and K. Standing (1981) Int. J. Mass Spectrom, Ion Phys. 40:185 
and Mamyrin et al. (1973) Sov. Phys. JETP 37:45, all incorporated herein 
by reference for all purposes. Briefly, the frequency tripled output of, 
e.g., a Q-switched Lumonics HY400 neodymium/yttrium aluminum garnet laser 
("Nd-YAG") (355 nm, 10-nsec output pulse) is focused by a lens (12-inch 
focal length) through a fused silica window onto a sample inside the mass 
spectrometer. The product ions formed by the laser are accelerated by a 
static electric potential of 30 kV. The ions then drift down a 2-m tube 
maintained at a vacuum of 30 .mu.Pa and their arrival at the end of the 
tube is detected and recorded using, e.g., a Lecroy TR8828D transient 
recorder. The transient records of up to 200 individual laser shots are 
summed together and the resulting histogram is plotted as a mass spectrum. 
Peak centroid determinations and data reduction can be performed using a 
VAX workstation or other computer system. 
Protein samples can be prepared for laser desorption analysis by the 
following procedure. The laser desorption matrix material that preferably 
absorbs above 300 nm is dissolved in aqueous 30% (vol/vol) acetonitrile 
containing 0.1% (wt/wt) trifluoroacetic acid, to make a standard solution 
at 20.degree. C. (.apprxeq.50 mM). A preferred matrix is 
.alpha.-cyano-4-hydroxy cinnamic acid. A solution containing the protein 
sample of interest is then added to the matrix solution to give a final 
protein concentration of 0.1-10 .mu.M. A small aliquot (0.5 .mu.l) of this 
mixture is then applied to a flat metal probe tip (2-mm diameter) and 
dried at room temperature with forced air. The resulting deposit is washed 
in 4.degree. C. distilled water by immersing the tip for 10 seconds. This 
washing step aids the removal of soluble ionic contaminants from the 
protein/matrix deposit, without removing the proteins or matrix. Once the 
sample is washed, it is inserted into the mass spectrometer and analyzed. 
The entire protocol, from the beginning of sample preparation to finished 
mass spectral analysis, takes about 15 minutes. 
Another method useful for determining the difference in mass of members of 
biopolymer ladder is acrylamide gel electrophoresis. 
EXAMPLE 
We determined the sequence of synthetic porcine renin substrate 
tetradecapeptide (Sigma) using carboxypeptidase Y. The peptide has the 
amino acid sequence DRVYIHPFHL LVYS SEQ ID NO:1! (using the one letter 
code for amino acid residues) and molecular weight of 1759.0 Da. FIG. 3 is 
the matrix-assisted laser desorption ionization mass spectrometric 
(MALDI-MS) spectrum of renin substrate tetradecapeptide. The MALDI-MS 
analysis was carried out on a laser desorption ionization time-of-flight 
mass spectrometer constructed at the Rockefeller University (R. Beavis and 
B. Chait (1990) Anal. Chem. 62:1836-1840). .alpha.-Cyano-4-hydroxycinnamic 
acid was used as the laser desorption matrix (Beavis et al. (1992) Org. 
Mass Spectrom. 27:156-158) in the form of saturated solution in 
water/acetonitrile (2:1, v/v). One .mu.l of peptide solution was mixed 
with 5 .mu.l of matrix solution. 0.5 .mu.l of sample solution was then 
applied on the sample probe and analyzed (FIG. 3). In this spectrum, the 
singly (MH+) and doubly (2+) protonated molecular ions were observed at 
m/z 1759.9 and 880.5 amu. The peak denoted as "d-Asp" (at m/z 1644.7 amu) 
is an amino-terminal truncated peptide impurity. 
The carboxy-terminal sequence of this peptide was obtained by using 
carboxypeptidase digestion in the presence of an excess amount of amino 
acid amide followed by or concurrently with matrix-assisted laser 
desorption ionization mass spectrometric data analysis (Hillenkamp et al. 
(1991) Anal. Chem. 63:1193-1202). The amino acid amide L-lysinamide was 
used to balance the fast digestion steps of the carboxypeptidase during 
the digestion and to eliminate the sequence gaps. The competitive 
carboxypeptidase Y sequencing reaction was carried out in the presence of 
an excess amount of lysinamide (1 M, L-Lysinamide dihydrochloride (Sigma)) 
at pH 7.5. 
Five .mu.l of peptide substrate (1 mM) and 5 .mu.l of carboxypeptidase Y 
(100 (g/ml) were incubated in the buffer solution (total volume 100 Al, 
consisting of 50 mM of Hepes, 5 mM of EDTA and 1M of lysinamide) at 
37.degree. C. for 20 minutes. The reaction was then stopped by adding 20 
.mu.l of 2% TFA to 10 .mu.l of reaction solution. One .mu.l reaction 
mixture solution was mixed with 5 .mu.l MALDI matrix and 0.5 .mu.l sample 
solution was analyzed by MALDI-MS. The resulting sequencing spectrum is 
shown in FIG. 4. The spectrum provides the carboxyl terminal sequence of 
up to seven amino acid residues of this polypeptide in a single operation. 
The amino acid sequence was determined as follows. The first 
carboxy-terminal amino acid residue was determined by adding 127 Da 
(L-Lysinamide residue mass) to the mass of the intact molecular ion (1760 
Da), to give 1887 Da, followed by subtraction of the highest mass of 
lysinamide modified peptide peak (1799.3 Da). This calculation gives a 
mass difference of 87.7 Da, which corresponds to a serine residue. The 
remaining carboxy-terminal sequence can be read out from the mass 
differences between adjacent peaks in the spectrum. The mass difference of 
126.9 Da between the peaks at m/z 1424.5 and 1297.6 corresponds to the 
lysinamide. The peaks on the lower mass side of the lysinamide residue 
correspond to the unmodified peptides and the peaks on the higher mass 
side of lysinamide correspond to the lysinamide modified peptides. 
For comparison, the same peptide was digested with carboxypeptidase Y in 
the absence of lysinamide. FIG. 5 shows the sequencing spectrum after 10 
minutes reaction at 37.degree. C. The spectrum contains several gaps which 
lack an intermediate member of the peptide ladder. These results clearly 
indicate that the method of this invention balances the fast hydrolysis 
reactions with the aminolysis reactions. As a result, the sequence gaps in 
the normal carboxypeptide Y digestion (FIG. 5) were filled up by the 
lysinamide modifications (FIG. 4). 
L-Lysinamide is used as a nucleophile and competitive substrate for peptide 
substrate hydrolysis by carboxypeptidase Y, which decreases the hydrolysis 
rate. The aminolysis product generated by formation of lysinamide at the 
C-terminus of peptide can slow down the hydrolysis rate. This is shown by 
the large amount of intact peptide remaining (FIG. 4). This reduced rate 
of hydrolysis is not a disadvantage. It provides for more control of the 
degradation/ladder-forming process. The rate can be increased by the 
simple addition of more enzyme. 
The present invention provides a substantially novel method for preparing 
biopolymer ladders. While specific examples have been provided, the above 
description is illustrative and not restrictive. Many variations of the 
invention will become apparent to those of skill in the art upon review of 
this specification. The scope of the invention should, therefore, be 
determined not with reference to the above description, but instead should 
be determined with reference to the appended claims along with their full 
scope of equivalents. 
All publications and patent documents cited in this application are 
incorporated by reference in their entirety for all purposes to the same 
extent as if each individual publication or patent document were so 
individually denoted. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 1 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AspArgValTyrIleHisProPheHisLeuLeuValTyrSer 
1510 
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