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Description  |
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This application claims the benefit of the filing date of PCT Application
US94/06064 filed May 27, 1994.
BACKGROUND OF THE INVENTION
This invention relates generally to methods and apparatus for desorption
and ionization of analytes for the purpose of subsequent scientific
analysis by such methods, for example, as mass spectrometry (MS) or
biosensors. Generally, analysis by mass spectrometry involves the
vaporization and ionization of a small sample of material, using a high
energy source, such as a laser, including a laser beam. The material is
vaporized from the surface of a probe tip into the gas or vapor phase by
the laser beam, and, in the process, some of the individual molecules are
ionized by the gain of a proton. The positively charged ionized molecules
are then accelerated through a short high voltage field and let fly
(drift) into a high vacuum chamber, at the far end of which they strike a
sensitive detector surface. Since the time-of-flight is a function of the
mass of the ionized molecule, the elapsed time between ionization and
impact can be used to determine the molecule's mass which, in turn, can be
used to identify the presence or absence of known molecules of specific
mass.
All known prior art procedures which present proteins or other large
biomolecules on a probe tip for laser desorption/ionization time-of-flight
mass spectrometry (TOF) rely on the preparation of a crystalline solid
mixture of the protein or other analyte molecule in a large molar excess
of acidic matrix material deposited on the bare surface of a metallic
probe tip. (The sample probe tip typically is metallic, either stainless
steel, nickel plated material or platinum). Embedding the analyte in such
a matrix was thought to be necessary in order to prevent the destruction
of analyte molecules by the laser beam. The laser beam strikes the solid
mixture on the probe tip and its energy is used to vaporize a small
portion of the matrix material along with some of the embedded analyte
molecules. Without the matrix, the analyte molecules are easily fragmented
by the laser energy, so that the mass, and identity, of the original
macromolecule is very difficult or impossible to determine.
This prior art procedure has several limitations which have prevented its
adaptation to automated protein or other macrobiological molecular
analysis. First, in a very crude sample it is necessary to partially
fractionate (or otherwise purify the sample as much as possible) to
eliminate the presence of excessive extraneous materials in the
matrix/analyte crystalline or solid mixture. The presence of large
quantities of components may depress the ion signal (either desorption,
ionization and/or detection) of the targeted analyte. Such purification is
time-consuming, expensive, typically results in low recovery (or complete
loss) of the analyte, and would be very difficult to do in an automated
analyzer.
Second, while the amount of analyte material needed for analysis by the
prior art method is not large (typically in a picomole range), in some
circumstances, such as tests on pediatric patients, analyte fluids are
available only in extremely small volumes (microliters) and may be needed
for performing several different analyses. Therefore, even the small
amount (i.e., volume) needed for preparation of the analyte/matrix
crystalline mixture for a single analysis may be significant. Also, only a
tiny fraction (a few thousandths or less) of analyte used in preparing the
solid analyte/matrix mixture for use on the probe tip is actually consumed
in the desorption or mass spectrometric analysis. Any improvement in the
prior art procedure which would make it possible to 1) use much less
analyte, 2) to locate the analyte or multiple analytes on the probe tip or
surface in a predetermined location, 3) to perform repeated analyses of
the same aliquot of analyte (e.g., before and after one or more chemical
and or enzymatic reactions), and 4) to conduct the test in a more
quantitative manner, would be highly advantageous in many clinical areas.
Third, the analyte protein, or other macromolecule, used in preparing the
solid solution of analyte/matrix for use on the probe tip is not suitable
for any subsequent chemical tests or procedures because it is bound up
(i.e., embedded) in the matrix material. Also, all of the matrix material
used to date is strongly acidic, so that it would adversely affect many
chemical reactions which might be attempted on the mixture in order to
modify the analyte molecules for subsequent examination. Any improvement
in the procedure which made it possible to conduct subsequent chemical
modifications or reactions on the analyte molecules, without removing them
from the matrix or the probe tip or without "matrix" altogether, would be
of enormous benefit to researchers and clinicians.
The first successful molecular mass measurements of intact peptides and
small proteins (only up to about 15 kDa) by any form of mass spectrometry
were made by bombarding surfaces with high energy particles (plasma
desorption and fast atom bombardment mass spectrometry); this breakthrough
came in 1981 and 1982. Improvements came in 1985 and 1986, however, yield
(signal intensities), sensitivity, precision, and mass accuracy remained
relatively low. Higher molecular mass proteins (about 20 to 25 kDa) were
not observed except on rare occasions; proteins representing average
molecular weights (approximately 70 kDa) were not ever observed with these
methods. Thus, evaluation of most proteins by mass spectrometry remains
unrealized.
In 1988, Hillenkamp and his coworkers used UV laser desorption
time-of-flight mass spectrometry and discovered that when proteins of
relatively high molecular mass were deposited on the probe tip in the
presence of a very large molar excess of an acidic, UV absorbing chemical
matrix (nicotinic acid) they could be desorbed in the intact state. This
new technique is called matrix-assisted laser desorption/ionization
(MALDI) time-of-flight mass spectrometry. Note that laser desorption
time-of-flight mass spectrometry (without the chemical matrix) had been
around for some time, however, there was little or no success determining
the molecular weights of large intact biopolymers such as proteins and
nucleic acids because they were fragmented (destroyed) upon desorption.
Thus, prior to the introduction of a chemical matrix, laser desorption
mass spectrometry was essentially useless for the detection of specific
changes in the mass of intact macromolecules. Note that the random
formation of matrix crystals and the random inclusion of analyte molecules
in the solid solution is prior art.
There are a number of problems and limitations with the prior art methods.
For example, previously, it has been found that it is difficult to wash
away contaminants present in analyte or matrix. Other problems include
formation of analyte-salt ion adducts, less than optimum solubility of
analyte in matrix, unknown location and concentration of analyte molecules
within the solid matrix, signal (molecular ion) suppression "poisoning"
due to simultaneous presence of multiple components, and selective analyte
desorption/ionization. Prior investigators, including Karas and Hillenkamp
have reported a variety of techniques for analyte detection using mass
spectroscopy, but these techniques suffered because of inherent
limitations in sensitivity and selectivity of the techniques, specifically
including limitations in detection of analytes in low volume,
undifferentiated samples. (Hillenkamp, Bordeaux Mass Spectrometry
Conference Report, pp. 354-62 (1988); Karas and Hillenkamp, Bordeaux Mass
Spectrometry Conference Report, pp. 416-17 (1988); Karas and Hillenkamp,
Analytical Chemistry, 60:2299 2301(1988); Karas, et al., Biomed. Environ.
Mass Spectrum 18:841-843 (1989).) The use of laser beams in time-of-flight
mass spectrometers is shown, for example, in U.S. Pat. Nos. 4,694,167;
4,686,366, 4,295,046, and 5,045,694, incorporated by reference.
The successful volatilization of high molecular weight biopolymers, without
fragmentation, has enabled a wide variety of biological macromolecules to
be analyzed by mass spectrometry. More importantly perhaps, it has
illustrated the potential of using mass spectrometry more creatively to
solve problems routinely encountered in biological research. Most recent
attention has been focused on the utility of matrix-assisted laser
desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS),
largely because it is rapid (min), sensitive (<pmol sample required), and
permits complex mixtures to be analyzed.
Although MALDI-TOF MS continues to be useful for the static
determination/verification of mass for individual analytes, in the case of
biopolymers, it is often differences in mass that provide the most
important information about unknown structures. Thus, for routine use in
structural biology, an unfortunate limitation of the MALDI-TOF MS
technique relates to sample preparation and presentation (deposition) on
an inert probe element surface, specifically, the requirement that
analytes be embedded (i.e., co-solidified) on the probe surface in a
freshly prepared matrix of crystalline organic acid. The random
distribution of analyte in a heterogeneous display of crystal matrix on
the probe element surface requires the deposition of far more analyte or
sample than is needed for the laser desorption process, even for the
collection of more than adequate mass spectra (e.g., multiple sets of 100
shots each). The remaining portion of the analyte is usually not recovered
for additional analyses or subsequent characterizations. Even though 1 to
10 pmol (sometimes less) of analyte are typically required for deposition
on the probe surface, it has been estimated that less than a few attomoles
are consumed during laser desorption. Thus, only 1 part in 10.sup.5 or
10.sup.6 of the applied analyte may be necessary; the rest is lost.
Another important loss of potential data associated with the embedding of
analyte in a solid matrix is the reduction or the complete elimination of
ability to perform subsequent chemical and/or enzymatic modifications to
the embedded analyte (e.g., protein or DNA) remaining on the probe
surface. Only another aliquot of analyte, or the ability to recover the
embedded analyte free of matrix (difficult with low recovery), allows what
we now refer to as differential mass spectrometry to be performed to
derive structural data.
In addition, there has been limited application of MS in biological fields,
likely due to the fact that many biologists and clinicians are intimidated
by MS and/or skeptical in regard to its usefulness. Further, MS is
perceived as inaccessible or too costly, particularly because SDS
polyacrylamide gel electrophoresis is an adequate substitute in some
instances where MALDI would be applied (e.g., separation of crude
biological fluids). In addition, MALDI has had little exposure in
biological and clinical journals.
SUMMARY OF THE INVENTION
An object of the invention is to provide improved methods, materials
composition and apparatus for coupled adsorption, desorption and
ionization of multiple or selected analytes into the gas (vapor) phase.
Another object is to provide a method and apparatus for affinity-directed
detection of analytes, including desorption and ionization of analytes in
which the analyte is not dispersed in a matrix solution or crystalline
structure but is presented within, on or above an attached surface of
energy absorbing "matrix" material through molecular recognition events,
in a position where it is accessible and amenable to a wide variety of
chemical, physical and biological modification or recognition reactions.
Another object is to provide such a method and apparatus in which the
analyte material is chemically bound or physically adhered to a substrate
forming a probe tip sample presenting surface.
A further object is to provide means for the modification of sample
presenting surfaces with energy-absorbing molecules to enable the
successful desorption of analyte molecules without the addition of
exogenous matrix molecules as in prior art.
A further object is to provide the appropriate density of energy-absorbing
molecules bonded (covalently or noncovalently) in a variety of geometries
such that mono layers and multiple layers of attached energy-absorbing
molecules are used to facilitate the desorption of analyte molecules of
varying masses.
A further object is to provide multiple combinations of surfaces modified
with energy-absorbing molecules, affinity-directed analyte capture
devices, phototubes, etc.
An additional object is to provide such a method and apparatus in which the
substrate forming the probe tip or other sample presenting surface is
derivatized with one or more affinity reagents (a variety of densities and
degrees of amplification) for selective bonding with predetermined
analytes or classes of analytes.
A further object is to provide such a system in which the affinity reagent
chemically bonds or biologically adheres to the target analyte or class of
analytes.
A still further object is to provide a method and apparatus for desorption
and ionization of analytes in which unused portion of the analytes
contained on the presenting surface remain chemically accessible, so that
a series of chemical, enzymatic or physical treatments of the analyte may
be conducted, followed by sequential analyses of the modified analyte.
A further object is to provide a method and apparatus for the combined
chemical or enzymatic modifications of target analytes for the purpose of
elucidating primary, secondary, tertiary, or quaternary structure of the
analyte and its components.
Another object is to provide a method and apparatus for desorption and
ionization of analyte materials in which cations other than protons
(H.sup.+) are utilized for ionization of analyte macromolecules.
Thus, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, an apparatus for measuring the mass
of an analyte molecule of an analyte sample by means of mass spectrometry,
said apparatus comprising a spectrometer tube; a vacuum means for applying
a vacuum to the interior of said tube; electrical potential means within
the tube for applying an accelerating electrical potential to desorbed
analyte molecules from said analyte sample; sample presenting means
removably insertable into said spectrometer tube, for presenting said
analyte sample in association with surface associated molecule for
promoting desorption and ionization of said analyte molecules, wherein
said surface molecule is selected from the group consisting of energy
absorbing molecule, affinity capture device, photolabile attachment
molecule and combination thereof; an analyte sample deposited on said
sample presenting means in association with said surface associated
molecules, whereby at least a portion of said analyte molecules not
consumed in said mass spectrometry analysis will remain accessible for
subsequent chemical, biological or physical analytical procedures; laser
beam means for producing a laser beam directed to said analyte sample for
imparting sufficient energy to desorb and ionize a portion of said analyte
molecules from said analyte sample; and detector means associated with
said spectrometer tube for detecting the impact of accelerated ionized
analyte molecules thereon.
In addition, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, a method in mass spectrometry to
measure the mass of an analyte molecule, said method comprising the steps
of: derivitizing a sample presenting surface on a probe tip face with an
affinity capture device having means for binding with an analyte molecule;
exposing said derivitized probe tip face to a source of said analyte
molecule so as to bind said analyte molecule thereto; placing the
derivitized probe tip with said analyte molecules bound thereto into one
end of a time-of-flight mass spectrometer and applying a vacuum and an
electric field to form an accelerating potential within the spectrometer;
striking at least a portion of the analyte molecules bound to said
derivitized probe tip face within the spectrometer with one or more laser
pulses in order to desorb ions of said analyte molecules from said tip;
detecting the mass of the ions by their time of flight within said mass
spectrometer; and displaying such detected mass.
Further, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, a method of measuring the mass of
analyte molecules by means of laser desorption/ionization, time-of-flight
mass spectrometry in which an energy absorbing material is used in
conjunction with said analyte molecules for facilitating desorption and
ionization of the analyte molecules, wherein the improvement comprises
presenting the analyte molecules on or above the surface of the energy
absorbing material, wherein at least a portion of the analyte molecules
not desorbed in said mass spectrometry analysis remain chemically
accessible for subsequent analytical procedures.
Additionally, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, an apparatus for facilitating
desorption and ionization of analyte molecules, said apparatus comprising:
a sample presenting surface; and surface associated molecules, wherein
said surface associated molecules are selected from the group consisting
of energy absorbing molecule, affinity capture device, photolabile
attachment molecule and combination thereof, said surface associated
molecules associated with said sample presenting surface and having means
for binding with said analyte molecules.
Further, there is provided a method for capturing analyte molecules on a
sample presenting surface and desorbing/ionizing said captured analyte
molecules from said sample presenting surface for subsequent analysis,
said method comprising: derivitizing said sample presenting surface with
an affinity capture device or photolabile attachment molecule having means
for binding with said analyte molecules; exposing said derivitized sample
present surface to a sample containing said analyte molecules; capturing
said analyte molecules on said derivitized sample presenting surface by
means of said affinity capture device or photolabile attachment molecule;
and exposing said analyte molecules, while bound to said derivitized
sample presenting surface by means of said affinity capture device or
photolabile attachment molecule, to an energy or light source to desorb at
least a portion of said analyte molecules from said surface.
Additionally, in accordance with the present invention, there is provided a
method for preparing a surface for presenting analyte molecules for
analysis, said method comprising: providing a substrate on said surface
for supporting said analyte; derivitizing said substrate with an affinity
capture device or photolabile attachment molecule having means for
selectively bonding with said analyte; and a means for detecting said
analyte molecules bonded with said affinity capture device or photolabile
attachment molecule.
Further, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, a sample probe for promoting
desorption of intact analytes into the gas phase comprising: a sample
presenting surface; and an energy absorbing molecule associated with said
sample presenting surface, wherein said sample probe promotes desorption
of an intact analyte molecule positioned on, above or between the energy
absorbing molecules when said sample probe is impinged by an energy
source. Further, the energy absorbing molecule in the probe is selected
from the group consisting of cinnamamide, cinnamyl bromide,
2,5-dihydroxybenzoic acid and .alpha.-cyano-4-hydroxycinnamic acid.
Additionally, in accomplishing the foregoing objects, there is provided in
accordance with the present invention, a sample probe for desorption of
intact analyte into the gas phase, comprising: a sample presentation
surface; and a surface associated molecule wherein said surface associated
molecule is a photolabile attachment molecule having at least two binding
sites, wherein at least one site is bound to the sample presentation
surface and at least one site is available to bind an analyte and wherein
the analyte binding site is photolabile.
In addition, in accomplishing the foregoing objects there is provided in
accordance with the present invention, a sample probe for promoting
desorption of intact analytes into the gas phase comprising: a sample
presentation surface; and either a mixture of at least two different
molecules selected from the group consisting of an affinity capture
device, an energy absorbing molecule and a photolabile attachment molecule
associated with said sample presentation surface; wherein when an analyte
is associated with said sample probe, said sample probe promotes the
transition of the analyte into the gas phase when said sample probe is
impinged by an energy source; or at least two different affinity capture
devices associated with said sample presentation surface; wherein, when
said sample probe is impinged by an energy source, said sample probe
promotes the transition of an analyte molecule into the gas phase at
different rates depending on the affinity capture device associated with
said analyte molecule.
In addition, in accomplishing the foregoing objects there is provided in
accordance with the present invention, a sample probe for promoting
desorption of intact analyte into the gas phase, comprising: a sample
presentation surface; and either a surface associated molecule, wherein
said surface associated molecule can function both as an energy absorbing
molecule and as an affinity capture device; or a surface associated
molecule wherein said surface associated molecule is a photolabile
attachment molecule having at least two binding sites, wherein at least
one site is bound to the sample presentation surface and at least one site
is available to bind an analyte and wherein the analyte binding site is
photolabile.
Additionally, there is provided in the present invention, a method in mass
spectrometry to measure the mass of an analyte molecule, said method
comprising the steps of: derivitizing a sample presenting surface on a
probe tip face with a photolabile attachment molecule (PAM), wherein said
PAM has at least two binding sites, one binding site binds to the sample
presenting surface and at least one binding site is available for binding
with an analyte molecule; exposing said derivitized probe tip face to a
source of said analyte molecule so as to bind said analyte molecule
thereto; placing the derivitized probe tip with said analyte molecules
bound thereto into one end of a time-of-flight mass spectrometer and
applying a vacuum and an electric field to form an accelerating potential
within the spectrometer; striking at least a portion of the analyte
molecules bound to said derivitized probe tip face within the spectrometer
with one or more laser pulses in order to desorb ions of said analyte
molecules from said tip; detecting the mass of the ions by their time of
flight within said mass spectrometer; and displaying such detected mass.
In addition, there is provided a method of measuring the mass of analyte
molecules by means of laser desorption/ionization, time-of-flight mass
spectrometry in which a photolabile attachment molecule (PAM) is used in
conjunction with said analyte molecules for facilitating desorption and
ionization of the analyte molecules, the improvement comprising:
presenting the analyte molecules on or above the surface of the PAM,
wherein at least a portion of the analyte molecules not desorbed in said
mass spectrometry analysis remain chemically accessible for subsequent
analytical procedures.
There is further provided in accordance with the present invention, a
sample probe for promoting of differential desorption of intact analyte
into the gas phase, comprising: a sample presentation surface; and at
least two different photolabile attachment molecules associated with said
sample presentation surface; wherein, when said sample probe is impinged
by an energy source, said sample probe promotes the transition of an
analyte molecule into the gas phase at different rates depending on the
photolabile attachment molecule associated with said analyte molecule.
Additionally, there is provided in accordance with the present invention, a
sample probe for promoting desorption of intact analytes into the gas
phase comprising: a sample presenting surface; and a photolabile
attachment molecule associated with said sample presenting surface;
wherein, when said sample probe is impinged by an energy source, said
sample probe promotes the transition of an intact analyte molecule into
the gas phase.
Further, there is provided in accordance with the present invention, a
method for biopolymer sequence determination comprising the steps of:
binding a biopolymer analyte to probe tip containing a sample presenting
surface having a surface selected molecule selected from the group
consisting of an energy absorbing molecule, an affinity capture device, a
photolabile attachment molecule and a combination thereof; desorption of
biopolymer analyte in mass spectrometry analysis, wherein at least a
portion of said biopolymer is not desorbed from the probe tip; analyzing
the results of the desorption modifying the biopolymer analyte still bound
to the probe tip; and repeating the desorption, analyzing and modifying
steps until the biopolymer is sequenced.
Other and further objects, features and advantages will be apparent and the
invention more readily understood from a reading of the following
specification and by reference to the accompanying drawings forming a part
thereof, wherein the examples of the presently preferred embodiments of
the invention are given for the purposes of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects and advantages of the invention will be
apparent from the following specification and from the accompanying
drawings.
FIG. 1 (upper profile) shows the mass spectrum of the three peptides (human
histidine rich glycoprotein metal-binding domains (GHHPH).sub.2 G (1206
Da), (GHHPH).sub.5 G (2904 Da), and human estrogen receptor dimerization
domain (D473-L525) (6168.4 Da)) desorbed in the presence of neutralized
energy absorbing molecules (sinapinic acid, pH 6.2). FIG. 1 (lower
profile) shows the sequential in situ metal (Cu)-binding of the peptides
in the presence of neutral energy absorbing molecules.
FIG. 2 (top profile) shows the mass spectrum of the human casein
phosphopeptide (5P, 2488 Da) desorbed in the presence of neutralized
energy absorbing molecules (sinapinic acid, pH 6.5). FIG. 2 (second from
top profile) shows the sequential in situ 5 min alkaline phosphatase
digestion to remove phosphate groups from the phosphopeptide. FIG. 2
(third from top profile) shows the mass spectrum of the phosphopeptide
after further in situ digestion (10 min) with alkaline phosphatase. FIG. 2
(bottom profile) shows the mass spectrum of the phosphopeptide after in
situ 10 min alkaline phosphatase digestion in the presence of acidic
energy absorbing molecules (2,5 dihydroxybenzoic acid, pH 2) as described
in prior art.
FIG. 3 shows a composite mass spectra of the (GHHPH).sub.5 G peptide (2904
Da) before (lower profile) and after (upper profile) in situ digestion by
carboxypeptidase P in the presence of neutralized energy absorbing
molecules (sinapinic acid, pH 6.2).
FIG. 4 shows a composite matrix-assisted laser desorption mass spectra of
peptide mixtures desorbed from solid glass, polypropylene-coated stainless
steel, polystyrene-coated stainless steel and solid nylon probe elements.
SEAC
FIG. 5, top profile shows the mass spectrum of sperm activating factor (933
Da) and neurotensin (1655 Da) (and their multiple Na-adducts) in the
peptide solution unadsorbed by the IDA-Cu(II) surface. FIG. 5, middle
profile, shows the mass spectrum of angiotensin I (1296.5 Da) plus
Na-adduct peaks that were selectively adsorbed on the IDA-Cu(II) surface.
FIG. 5, bottom profile, and FIG. 6, bottom profile, show the mass spectrum
of the same angiotensin I adsorbed on IDA-Cu(II) after water wash. FIG. 6,
middle profile, shows the sequential in situ copper-binding (1 and 2 Cu)
by affinity adsorbed angiotensin I. FIG. 6, top profile, shows the
sequential in situ trypsin digestion of the affinity adsorbed angiotensin
I.
FIG. 7 shows the mass spectrum of myoglobin (4 to 8 fmole) affinity
adsorbed on IDA-Cu(II) surface.
FIG. 8 (top profile) shows the mass spectrum of synthetic casein peptide
(1934 Da) with multiple phosphorylated forms affinity adsorbed from a
crude mixture on TED-Fe(III) surface. After sequential in situ alkaline
phosphatase digestion, only the original nonphosphorylated form remained
(lower profile).
FIG. 9, bottom profile, shows the mass spectrum of total proteins in infant
formula. FIG. 9, second to the bottom profile, shows the mass spectrum of
phosphopeptides in infant formula affinity adsorbed on TED-Fe(III)
surface. FIG. 9, third from the bottom profile, shows the mass spectrum of
total proteins in gastric aspirate of preterm infant obtained after
feeding the infant formula. FIG. 9, top profile, shows the mass spectrum
of phosphopeptides in the gastric aspirate affinity adsorbed on
TED-Fe(III) surface.
FIG. 10A shows the composite mass spectra of human and bovine
histidine-rich glycoprotein adsorbed on IDA-Cu(II) surface before and
after N-glycanase digestion. The mass shifts represent the removal of
carbohydrate from the respective glycoproteins. FIG. 10B shows the
composite mass spectra of trypsin digested peptides from the
deglycosylated proteins of the two species (top profile for human protein,
second from bottom profile for bovine protein) and in situ Cu(II)-binding
of the trypsin digested peptides of the two species (second from top
profile for human protein, bottom profile for bovine protein; the numbers
1, 2 indicate the number of copper bound). FIG. 10C shows that one such
Cu(II)-binding peptide (bottom profile) has at least 4 His residues which
are specifically modified by diethylpyrocarbonate to form 4
N-carbethoxy-histidyl adducts (1-4, top profile). FIG. 10D shows the
partial C-terminal sequence of the major Cu-binding peptide in the bovine
histidine rich glycoprotein.
FIG. 11 (bottom profile) shows the mass spectrum of rabbit anti-human
lactoferrin immunoglobulin alone (control) affinity adsorbed on sheep
anti-rabbit IgG paramagnetic surface. The top profile shows the mass
spectrum of human lactoferrin and rabbit anti-human lactoferrin
immunoglobulin complex affinity adsorbed on sheep anti-rabbit IgG
paramagnetic surface.
FIG. 12 shows the mass spectrum of human lactoferrin affinity adsorbed from
preterm infant urine on a anti-human lactoferrin immunoglobulin nylon
surface. FIG. 13 shows the equivalent mass spectrum of whole preterm
infant urine containing 1 nmole/ml of lactoferrin.
FIG. 14 (lower profile) shows the mass spectrum of pure bovine histidine
rich glycoprotein. The upper profile shows the mass spectrum of bovine
histidine rich glycoprotein and fragments affinity adsorbed from bovine
colostrum on anti-bovine histidine rich glycoprotein immunoglobulin
surface.
FIG. 15 shows the composite mass spectra of the peptides of follicle
stimulating hormone recognized by the different anti-follicle stimulating
hormone antibodies.
FIG. 16 shows the mass spectrum of human lactoferrin affinity adsorbed on a
single bead of single-stranded DNA agarose deposited on a 0.5 mm diameter
probe element.
FIG. 17 shows the mass spectrum of human lactoferrin affinity adsorbed from
preterm infant urine on single-stranded DNA surface
FIG. 18A shows the composite mass spectra of the total proteins in human
duodenal aspirate (lower profile) and the trypsin affinity adsorbed from
the aspirate on a soybean trypsin inhibitor surface (upper profile). FIG.
18B shows the mass spectrum of trypsin affinity adsorbed from 1 ul of
aspirate on a soybean trypsin inhibitor nylon surface.
FIG. 19A shows the mass spectrum of biotinylated insulin affinity adsorbed
from human urine on a Streptavidin surface. FIG. 19B shows the mass
spectrum of biotinylated insulin affinity adsorbed from human plasma on a
Streptavidin surface.
FIG. 20 (upper profile) shows the mass spectrum of total proteins in human
serum. FIG. 20 (lower profile) shows the mass spectrum of serum albumin
affinity adsorbed from human serum on a Cibacron-blue surface.
SEND
FIG. 21 shows the molecular structure of surface bound cinnamamide; R
represents the surface plus cross-linker.
FIG. 22 (upper profile) shows the mass spectrum of peptide mixtures
desorbed from surface bound cinnamamide. FIG. 20 (lower profile) shows the
mass spectrum of the same peptide mixtures with free cinnamamide.
FIG. 23 shows the molecular structure of surface bound cinnamyl bromide; R
represents the surface plus cross-linker.
FIG. 24 (upper profile) shows the mass spectrum of peptide mixtures
desorbed from surface bound cinnamyl bromide. FIG. 22 (lower profile)
shows the mass spectrum of the same peptide mixtures with free cinnamyl
bromide.
FIG. 25 shows the molecular structure of surface bound MAP-dihydroxybenzoic
acid; R represents the surface plus cross-linker.
FIG. 26 (upper profile) shows the mass spectrum of peptide mixtures
desorbed from surface bound MAP alone. FIG. 26 (lower profile) shows the
mass spectrum of the same peptide mixtures desorbed from surface bound
MAP-dihydroxybenzoic acid.
FIG. 27A shows the mass spectrum (1,200-50,000 m/z region) of myoglobin
desorbed from surface bound .alpha.-cyano-4-hydroxycinnamic acid. FIG. 27B
shows the same mass spectrum in the low mass region (0-1200 m/z).
FIG. 28 shows the molecular structure of energy absorbing molecules bound
to polyacrylamide or nylon or acrylic surface via glutaraldehyde
activation.
FIG. 29 shows the molecular structure of energy absorbing molecules bound
to polyacrylamide or nylon or acrylic surface via divinyl sulfone
activation.
FIG. 30 shows the molecular structure of energy absorbing molecules bound
to polyacrylamide or nylon or acrylic surface via dicyclohexylcarbodiimide
activation.
FIG. 31 shows the molecular structure of energy absorbing molecules bound
to polyacrylamide or nylon or acrylic surface with multiple antigenic
peptide via dicyclohexylcarbodiimide activation.
FIG. 32 shows the molecular structure of thiosalicylic acid bound to
iminodiacetate (IDA)-Cu(II) surface.
FIG. 33 shows the mass spectrum of human estrogen receptor dimerization
domain desorbed from thiosalicylic acid-IDA-Cu(II) surface.
FIG. 34 shows the molecular structure of .alpha.-cyano-4-hydroxycinnamic
acid bound to DEAE surface.
FIG. 35(A) shows the mass spectrum of human estrogen receptor dimerization
domain desorbed from sinapinic acid-DEAE surface. FIG. 35B shows the mass
spectrum of myoglobin desorbed from .alpha.-cyano-4-hydroxycinnamic acid
DEAE surface.
FIG. 36 shows the molecular structure of .alpha.-cyano-4-hydroxycinnamic
acid bound to polystyrene surface.
SEPAR
FIG. 37 shows the C-terminal sequence analysis of surface immobilized via
photolytic bond histidine rich glycoprotein metal binding domain.
DETAILED DESCRIPTION OF THE INVENTION
It will be apparent to one skilled in the | | |
