U.S. patent application number 14/583376 was filed with the patent office on 2015-05-07 for methods for isotopically labeling biomolecules using mammalian cell-free extracts.
This patent application is currently assigned to PIERCE BIOTECHNOLOGY, INC.. The applicant listed for this patent is Derek Karl Baerenwald, Ryan D. Bomgarden, Eric Leigh Hommema, Penny JoAnn Jensen, John Charles Rogers. Invention is credited to Derek Karl Baerenwald, Ryan D. Bomgarden, Eric Leigh Hommema, Penny JoAnn Jensen, John Charles Rogers.
Application Number | 20150125875 14/583376 |
Document ID | / |
Family ID | 47627169 |
Filed Date | 2015-05-07 |
United States Patent
Application |
20150125875 |
Kind Code |
A1 |
Bomgarden; Ryan D. ; et
al. |
May 7, 2015 |
METHODS FOR ISOTOPICALLY LABELING BIOMOLECULES USING MAMMALIAN
CELL-FREE EXTRACTS
Abstract
Methods for producing an isotope-labeled mammalian, including a
human, biomolecule, such as polypeptides and proteins, in a
cell-free protein synthesis system. A biomolecule standard is
produced having at least one isotope different in abundance than
that of the naturally occurring isotopes in the biomolecule.
Methods for quantifying biomolecules standards expressed using
mammalian cell-free extracts are disclosed. Methods for producing
such standards, kits, systems and reagents, relating to the use of
isotope-labeled biomolecule as quantification standards in mass
spectrometric and nuclear magnetic resonance analysis.
Inventors: |
Bomgarden; Ryan D.;
(Winnebago, IL) ; Hommema; Eric Leigh; (Roscoe,
IL) ; Rogers; John Charles; (Rockton, IL) ;
Jensen; Penny JoAnn; (Marengo, IL) ; Baerenwald;
Derek Karl; (Minneapolis, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bomgarden; Ryan D.
Hommema; Eric Leigh
Rogers; John Charles
Jensen; Penny JoAnn
Baerenwald; Derek Karl |
Winnebago
Roscoe
Rockton
Marengo
Minneapolis |
IL
IL
IL
IL
MN |
US
US
US
US
US |
|
|
Assignee: |
PIERCE BIOTECHNOLOGY, INC.
Rockford
IL
|
Family ID: |
47627169 |
Appl. No.: |
14/583376 |
Filed: |
December 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13565135 |
Aug 2, 2012 |
8945861 |
|
|
14583376 |
|
|
|
|
61514695 |
Aug 3, 2011 |
|
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Current U.S.
Class: |
435/7.1 ; 435/23;
435/40.5 |
Current CPC
Class: |
G01N 2560/00 20130101;
C12P 21/02 20130101; G01N 33/58 20130101; G01N 33/6848 20130101;
G01N 2458/15 20130101 |
Class at
Publication: |
435/7.1 ; 435/23;
435/40.5 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/58 20060101 G01N033/58 |
Claims
1. A method for quantitating a protein analyte in a sample, the
method comprising preparing a heavy isotope labeled protein,
providing a known quantity of the heavy isotope labeled protein to
the sample to create a mixture, where the labeled protein and the
protein analyte comprise at least one portion of sequence identity,
subjecting the mixture to digestion conditions for preparation of
resultant peptide fragments, where the digestion results in at
least a first labeled peptide fragment and a first peptide
fragment, both having the same sequence, subjecting the prepared
mixture to mass spectrometry, and quantitating the amount of the
analyte in the sample using the known amount of the first labeled
peptide fragment.
2. The method of claim 1, wherein the heavy isotope labeled protein
is a standard and provides at least one of absolute and relative
quantitation of the analyte.
3. The method of claim 1, wherein the heavy isotope labeled protein
is purified prior to providing to the sample.
4. The method of claim 3, wherein the heavy isotope labeled protein
is quantitated either before or after purification.
5. The method of claim 1, wherein the heavy isotope labeled
protein, which is provided to the sample prior to digestion,
provides a means to assess digestion efficiency.
6. The method of claim 1, wherein the heavy isotope labeled protein
is serially diluted before providing to the sample.
7. The method of claim 6, wherein the serially diluted heavy
isotope labeled protein is used to generate a standard curve, which
allows for absolute quantitation of the analyte.
8. The method of claim 1, wherein the heavy isotope labeled protein
is produced either by in vitro translation or in vitro
transcription/translation using a mammalian cell-free extract.
9. The method of claim 8, wherein the mammalian cell-free extract
is prepared from cells cultured in media containing heavy
isotope-labeled amino acids.
10. The method of claim 1, wherein the heavy isotope is an isotope
different in abundance than the naturally occurring isotope.
11. The method of claim 1, wherein a plurality of heavy isotope
labeled proteins is provided to the sample.
12. A kit comprising a heavy isotope labeled protein, and
instructions for using the heavy isotope labeled protein as an
internal standard for mass spectrometry.
Description
[0001] This application is a Continuation of co-pending U.S.
application Ser. No. 13/565,135 filed Aug. 2, 2012, which claims
priority to U.S. application Ser. No. 61/514,695 filed Aug. 3,
2011, each of which is expressly incorporated by reference herein
in its entirety.
[0002] Methods to produce an isotope-labeled protein and/or
polypeptide in a cell-free in vitro protein synthesis system, i.e.,
either an in vitro translation system (IVT) or an in vitro coupled
transcription/translation system (IVTT); the abbreviations are used
interchangeably unless distinguished. A method for quantifying
isotope-labeled protein and/or polypeptide standards for use as a
relative or absolute standard in mass spectrometry (MS) analysis. A
method for producing stable-isotope labeled proteins for nuclear
magnetic resonance (NMR) analysis. A reagent kit for protein and/or
polypeptide synthesis for performing cell-free in vitro protein
synthesis and for quantifying isotope-labeled protein standards for
use in MS and/or NMR.
[0003] Proteomics uses methods such as Western Blotting, enzyme
linked immunosorbent assays (ELISA), NMR, and MS to understand
protein structure, function, and interactions. MS can
simultaneously identify proteins and protein post-translational
modifications (PTM), but MS protein quantification requires the use
of protein standards. One method for protein standard expression is
in vitro coupled transcription/translation (IVT), in which a
cellular extract system transcribes DNA into mRNA, which is
subsequently translated into protein. Most IVT systems utilize
prokaryotic (bacteria) or non-human eukaryotic (wheat germ or
rabbit reticulocyte) cell extracts. These systems lack components
needed for proper protein folding and proper post-translational
modifications to result in biologically active human proteins.
[0004] MS is a powerful method to elucidate the composition of
complex proteomic samples. Proteomic analysis has traditionally
involved isolating proteins from biological samples, followed by
fractionating the proteins using one- or two-dimensional gel
electrophoresis. Fractionated proteins are subsequently analyzed by
MS, either intact proteins using "top down" methods, or proteins
that have been enzymatically digested into peptides in "bottom up"
analysis. Relative differences between samples can be measured
semi-quantitatively, either by assessing gel band or spot staining
intensity, or by the number of MS spectra. These label-free methods
are, however, highly variable due to differences in peptide
ionization efficiency. In addition, proteomic samples typically
have a large dynamic range of protein abundance with high
heterogeneity, resulting in significant under-sampling using
gel-based analysis and MS instruments with lower sensitivity and
duty cycle.
[0005] Isotopically-labeled internal standards are required to
accurately quantify differences in samples analyzed by MS. If the
internal standard concentration is known, isotopically-labeled
internal standards permit a standard curve to be generated to
determine the absolute concentration of analyte in the sample.
Internal standards for MS analysis ideally are biologically and
chemically indistinguishable from the analyte to be measured.
[0006] Isotopes have traditionally been incorporated into peptide
and protein standards by numerous chemical, enzymatic, and
metabolic labeling methods. One common labeling method uses
chemically synthesized isotope-labeled peptides for absolute
quantitation, i.e., AQUA method. The AQUA method introduces known
quantities of isotope-labeled peptides into biological samples to
be analyzed, permitting the relative quantification of unlabeled
peptides. Absolute quantitation can be accomplished by classic
isotope dilution measurements, where stable isotope-labeled
peptides are used to generate a standard curve.
[0007] Alternatively, peptides in a sample can be labeled using
isotopic or isobaric chemical tags, e.g., isotope dimethylation,
iCAT, iTRAQ or TMT reagents to create internal reference peptide
standards for relative quantitation. These methods conjugate and/or
covalently attach chemical tags to peptides and/or proteins.
[0008] Enzymatic methods for isotope labeling generally add
.sup.18O isotopes to peptide carboxyl termini through tryptic
digestion in .sup.18O-labeled water. Stable isotopes can be
metabolically incorporated into proteins in cell culture (stable
isotope cell culture, SILAC). SILAC methods use metabolic
incorporation into proteins of heavy isotope-labeled amino acids or
non-heavy isotope-labeled, i.e., unlabeled or light, amino acids.
Heavy isotopes that can be used are stable isotopes such as, but
not limited to, .sup.13C, .sup.15N, .sup.74Se, .sup.76Se,
.sup.77Se, .sup.78Se, .sup.82Se, .sup.18O, and .sup.2H. An example
of the SILAC technique used for metabolic incorporation of isotopes
uses Escherichia coli (E. coli) cultured with media supplemented
with heavy isotope-labeled amino acids to express isotope-labeled
proteins or concatenated polypeptides (QConCat).
[0009] Although both peptide and protein isotope-labeled standards
are applicable for relative and absolute MS quantitation, protein
standards have benefits over peptide standards. In a majority of MS
sample preparation workflows, proteins are extracted from cells or
tissues before fractionation and enzymatic digestion. Protein
standards can be added to samples immediately after cell lysis and
before any additional sample preparation, which results in less
variance from sample processing. Protein standards provide an added
control for enzymatic digestion efficiency. Upon digestion, protein
standards also provide multiple peptides that can be used for
quantitation without prior knowledge of which peptide may give the
best MS signal. Depending on the protein source, protein standards
may also contain post-translational modifications that can be
monitored.
[0010] Current methods to isotopically label proteins are limited.
SILAC labeling incorporates heavy isotope-labeled amino acids into
proteins; however, it has disadvantages for making routine protein
standards. Because SILAC is an in vivo metabolic method that labels
all proteins, it is costly and wasteful for expression of
individual isotopically-labeled protein standards. Although SILAC
is amenable to whole organism labeling (e.g. bacteria, yeast,
worms, mice), human protein standards are limited to those
expressed in tissue cultured cell lines. Expression of recombinant
proteins in SILAC cells has been used to increase the diversity of
proteins that can be labeled by SILAC; however, many proteins that
are toxic or result in cell cycle arrest or apoptosis cannot be
expressed in vivo. Isotope scrambling of amino acids has been
observed for many cell lines used for SILAC. This phenomenon has
been shown to be caused by heavy isotope-labeled amino acid
catabolism, and is especially common for heavy isotope-labeled
arginine, which can be converted to heavy isotope-labeled proline
by arginine dehydrogenases.
[0011] In vitro expression using cell-free extracts is another
method to generate isotope-labeled proteins. This method allows
expression of a variety of recombinant proteins, including many
that cannot be expressed in vivo. Cell-free expression typically
does not suffer from isotope scrambling observed during in vivo
SILAC methods because stable isotope incorporation can be limited
to recombinant proteins of interest and takes less than 24 hours.
Two cell-free systems used for isotopic labeling of protein are E.
coli and wheat germ extracts; both systems express protein
standards with isotope incorporation up to 95% into only the
expressed recombinant protein. Although E. coli and wheat germ
extracts can be scaled to express moderate amounts, e.g., 0.1 mg-10
mg of protein standard, they are inadequate for expressing many
mammalian proteins. Both systems are derived from simple organisms
that have different amino acid codon usage compared to complex
mammalian genomes, so expression constructs may have to be codon
optimized to maximize expression in these systems. Endogenous
chaperones in these systems are not suited for the correct folding
of mammalian proteins, leading to mis-folded proteins and protein
aggregation. Proteins expressed in E. coli and wheat germ extracts
are typically missing normal post-translational modifications,
including phosphorylation and glycosylation, which may be required
for protein function or protein-protein interactions. These
features may be important to generate an isotopically-labeled
protein standard that is the most biologically equivalent to the
proteins of mammalian proteomes.
[0012] Peptide standards remain the predominant standard used for
relative and absolute quantitation, despite advantages of protein
standards over peptide standards. The main reason for this is due
to a lack of isotopically-labeled standards for all proteins, where
peptides can be readily chemically synthesized. Protein standards
are difficult to quantify because they typically must first be
purified to homogeneity and then measured using either absorbance
at 280 nm (A.sub.280) or a protein assay. Both techniques have high
protein to protein variability. A.sub.280 measurements require a
known molar extinction coefficient unique to each protein or
protein isoform to determine protein concentration. Protein assays
such as Bradford, Lowry, and bicinchoninic acid (BCA) have been
used to quantify purified proteins, but can give widely different
results depending on the amino acid composition of the protein. In
contrast, peptide standards can be readily measured using small
amounts of peptide by amino acid analysis (AAA) to determine
peptide composition and concentration.
[0013] Stable, isotopically-labeled protein standards represent the
"gold standard" for targeted MS proteomics quantitation. Heavy
labeled standards are required for clinically validated assays, but
production of heavy proteins is costly and most widely demonstrated
through in vivo SILAC methods. A human-cell derived in vitro
protein expression system is available (Pierce Biotechnologies,
Rockford Ill.) that produces higher yields of expressed proteins
and improves post-translational modifications such as
phosphorylation and glycosylation compared to other eukaryotic and
prokaryotic systems. Because proteins produced using this system
are more biologically equivalent to the proteins of mammalian
proteomes, the disclosed system can improve isotopically-labeled
mammalian protein standards if heavy labeled components such as
amino acids or tRNAs could be introduced to replace or compete out
endogenous light components of the lysate.
[0014] A human-cell derived in vitro protein expression system is
disclosed. This system provided higher yields of expressed proteins
and significant improvement of post-translational modifications,
such as phosphorylation and glycosylation, compared to other
eukaryotic and prokaryotic systems. Proteins produced using the
disclosed method were more biologically equivalent to the proteins
of mammalian proteomes compared to other methods. The disclosed
method was used to make improved isotopically-labeled protein
standards by introducing heavy labeled components, such as amino
acids and/or tRNAs, to replace or compete with endogenous non-heavy
labeled components of the lysate, also termed light components of
the lysate.
[0015] Using a cell lysate that was derived from SILAC-labeled HeLa
cells, having a high probability of producing an
isotopically-labeled protein, endogenous amino acids (L-lysine or
L-arginine) and tRNAs linked to these amino acids were assumed to
be isotopically labeled after 4-5 cell doublings of cells grown in
media containing only .sup.13C.sub.6.sup.15N.sub.2 L-lysine-2HCl
and/or .sup.13C.sub.6.sup.15N.sub.4 L-arginine-HCl. Upon
verification that all proteins in the HeLa cells were >98%
labeled using SILAC, a lysate was prepared using standard methods
developed for a coupled in vitro transcription/translation (IVT)
system. Test expression of a model protein, histidine-tagged green
fluorescent protein (HIS-tagged GFP), confirmed that the heavy
isotope-labeled lysate was capable of protein expression; however,
protein expression yields were about 5-fold less than control
unlabeled lysates. This result was reproducible, appeared to be a
consequence of poorer cell health before lysate production, and may
be linked to use of dialyzed fetal bovine serum (FBS) required for
the SILAC method. Despite the low yield of HIS-tagged GFP
expression using heavy isotope-labeled lysates, the average isotope
incorporation for GFP protein, based on MS of tryptic peptides, was
>95% when protein was expressed using extracts supplemented with
additional heavy isotope-labeled amino acids and when exogenous
tRNAs were omitted.
[0016] These results demonstrated production of a heavy
isotope-labeled protein using a costly and inefficient human IVT
system: the entire lysate, instead of only the expressed protein,
was heavy isotope-labeled while labeling of only the expressed
protein is required for production of a protein standard. Using as
controls heavy lysates supplemented with light amino acids, and
normal light lysates supplemented with heavy amino acids, about
30-40% isotope was incorporated for both controls after MS analysis
of HIS-tagged GFP. Omission of exogenous tRNAs increased isotope
incorporation by an additional 10%, to a final average
incorporation of 50%. These levels of incorporation were not
expected, because traditional prokaryotic and eukaryotic IVT
systems typically have <5% isotopically-labeled amino acid
incorporation unless they are modified. A published modification
method removed endogenous free amino acids by desalting or
dialyzing the lysates. Another used ion exchange chromatography to
remove endogenous tRNAs, which may be charged with light amino
acids. However, this tRNA-depleted lysate must be supplemented with
exogenous tRNAs and amino acids for protein synthesis.
[0017] By titrating the amount of heavy amino acids added to light
lysates, the isotope incorporation efficiency of expressed
HIS-tagged GFP was increased to >95% at heavy amino acid
(.sup.13C.sub.6.sup.15N.sub.2 L-lysine-2HCl and/or
.sup.13C.sub.6.sup.15N.sub.4L-arginine-HCl) final concentrations
>1 mM. This result was not expected; the concentration of the
amino acid supplement was 2 mM and was believed not to be limiting,
i.e., it was in excess, for production of proteins in the disclosed
coupled-IVT system. The same results from HIS-tagged GFP were
obtained using amino acid titration with another protein,
hemagglutinin (HA)-tagged Bcl-2-associated death promoter
(BAD).
[0018] Using the disclosed methods, the following proteins were
expressed with isotope incorporation efficiencies >95%: AKT1,
tumor protein 53 (TP53), glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), parathyroid hormone (PTH), retinoblastoma (RB), human
chorionic gonadotropin (hGC.beta.), cyclin D1 (CCND1) and
3-phosphoinositide dependent protein kinase-1 (PDPK1).
Supplementing the reactions with light amino acid mixture absent
lysine and/or arginine, termed a drop out mixture, was not
necessary for robust protein expression or for heavy isotope
incorporation. The disclosed method was less expensive than using a
heavy labeled-isotope labeled lysate. The disclosed method was
demonstrated for over ten different proteins.
[0019] During purification of over-expressed heavy HA-BAD protein,
two additional proteins co-purified. Using MS, these proteins were
determined to be 14-3-3 proteins, which are known to form a
heterodimeric complex with phosphorylated BAD and which have
numerous sites of modification including sites known to be required
for 14-3-3 protein binding. These findings demonstrated that the
proteins that were expressed using the inventive heavy IVT system
were functional; they were modified and integrated into protein
complexes.
[0020] Human PTH was expressed using the inventive method as an
internal standard (spike) for a MS immunoprecipiation assay. A
typical yield of heavy isotope-labeled proteins expressed using the
coupled human IVT or IVTT systems is 40 .mu.g/ml to 80 .mu.g/ml in
8-10 hours. In contrast, a high yield-coupled-IVT or IVTT system
can produce >100 .mu.g/ml heavy proteins since protein
expression continues for up to 16 hr. Higher amounts of protein are
required for NMR protein structure applications. NMR typically
requires proteins with all carbons or nitrogens isotopically
labeled for analysis, i.e., total protein labeling of all amino
acids. The inventive high yield method is useful for MS structural
analysis, which typically requires 10 .mu.g-100 .mu.g purified
isotope-labeled protein. For a high-yield system, the IVT reaction
was performed in a dialysis chamber that was seated in a tube
containing additional small molecules (nucleotides, ATP, salts, and
amino acids) in a dialysate. In contrast to the smaller scale
coupled IVT reactions, high-yield expression required additional
amino acids.
[0021] A custom dialysate was prepared containing a light amino
acid mixture without L-lysine and L-arginine arginine to supplement
with L-lysine-2HCl and .sup.13C.sub.6.sup.15N.sub.4 L-arginine-HCl.
GFP and three different human proteins were expressed with GFP
heavy isotope incorporation determined to be >97% by MS
analysis, as shown in FIG. 6.
[0022] The disclosed method produced an isotope-labeled protein in
a mammalian cell-free expression system. The disclosed method
quantified isotope-labeled protein standards for use as a relative
or absolute standard with MS. The disclosed kit was used for
protein synthesis using the disclosed method, and the mammalian
proteins synthesized contained post-translational modifications,
such as glycosylation and phosphorylation, evidencing that the
method resulted in an intact, functional mammalian protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The patent or application file contains at least one drawing
executed in color. A Petition under 37 C.F.R. .sctn.1.84 requesting
acceptance of the color drawing is being filed separately. Copies
of this patent or patent application publication with color
drawing(s) will be provided by the Office upon request and payment
of the necessary fee.
[0024] FIGS. 1A and 1B schematically show heavy isotope-labeled
recombinant protein expression, purification, and mass spectroscopy
(MS) analysis.
[0025] FIGS. 2A-2D show isotope-labeled green fluorescent protein
(GFP) expression using human IVT extracts using different human in
vitro translation extracts, also referred to as lysates.
[0026] FIGS. 3A-3B schematically show a high-yield human coupled in
vitro transcription/translation expression of isotopically-labeled
GFP.
[0027] FIG. 4 shows linearity of MS quantification using a heavy
isotope-labeled protein standard, stable isotopically-labeled GFP,
as a standard for MS quantitation of a non-heavy-isotope labeled or
light GFP standard dilution.
[0028] FIGS. 5A-D show IVT expression of isotopically-labeled human
proteins.
[0029] FIGS. 6A-B show expression of a heavy isotope BAD protein
and MS analysis.
[0030] Methods and kits to produce and quantify
isotopically-labeled biomolecules, such as polypeptides and
proteins, for use as standards for relative and absolute
quantitation of samples by mass spectrometry (MS). A method for
producing isotope-labeled biomolecules, such as polypeptides and
proteins, using a mammalian cell-free protein synthesis system. One
embodiment is a method that produces a biomolecule standard having
at least one isotope different in abundance than that of the
naturally occurring isotopes in the biomolecule of interest. One
embodiment is a method that produces an isotope-labeled protein
using a mammalian cell-free extract, also termed a cell lysate. One
embodiment is a mammalian cell-free extract derived from human
cells.
[0031] One embodiment is an in vitro method for producing heavy
isotope-labeled proteins by translating an mRNA template, i.e., an
in vitro translation method. This embodiment combined a mammalian
cell-free extract with at least one mRNA, a plurality of accessory
expression proteins, an ATP regeneration system, and heavy
isotope-labeled amino acids to form an in vitro translation system,
which is incubated under conditions to result in production of at
least one heavy isotope-labeled protein from the mRNA.
[0032] One embodiment is an in vitro method for producing heavy
isotope-labeled protein by transcribing a DNA template and then
translating the resultant mRNA, i.e., a coupled in vitro
transcription/translation method. This embodiment combined a
mammalian cell-free extract with at least one DNA, at least one RNA
polymerase, a plurality of accessory expression proteins, an ATP
regeneration system, and a plurality of heavy isotope-labeled amino
acids and/or heavy isotope-labeled tRNAs to form an in vitro
transcription/translation system, which was incubated under
conditions to result in production of at least one heavy
isotope-labeled protein from the DNA. To increase efficiency of
translation, in one embodiment the mammalian cell-free extract was
preincubated for at least one minute with at least one accessory
protein and a plurality of heavy isotope-labeled amino acids and/or
heavy isotope-labeled tRNAs. Then, and without removing any
component, DNA, RNA polymerase, and an ATP regeneration system were
added to form an in vitro transcription/translation system, which
was incubated under conditions to result in production of at least
one heavy isotope-labeled protein from the DNA. This method of
preincubation of the accessory proteins with the mammalian
cell-free extract and the subsequent addition of heavy amino acids
and DNA has been shown in the art to prevent phosphorylation of
eukaryotic translation initiation factor 2A (EIF2A). This is
necessary for high level IVT and IVTT protein expression using HeLa
cell extracts.
[0033] One embodiment is an in vitro method for producing heavy
isotope-labeled protein using a coupled transcription/translation
system. This embodiment combined a mammalian cell-free extract with
at least one DNA, at least RNA polymerase, an ATP regeneration
system, a plurality of non-heavy isotope-labeled amino acids and/or
non-heavy isotope-labeled tRNAs, and then added a plurality of
heavy isotope-labeled amino acids and/or heavy isotope-labeled
tRNAs in a concentration sufficient to replace the non-heavy
isotope-labeled amino acids and/or non-heavy isotope labeled tRNAs
to form an in vitro transcription/translation system, which was
incubated under conditions to result in production of at least one
heavy isotope-labeled protein from the DNA. This method resulted in
increased protein expression over time. Such methods supplementing
tRNAs charged with heavy amino acids increased isotope
incorporation efficiency to >50%, >95%, >97%, and >99%
in embodiments. Necessary reaction mix components present in the
dialysate continuously exchanged into the IVT reaction lysate
through the dialysis membrane, and potential translation inhibitory
compounds were diluted from the lysate by dialysis.
[0034] Accessory expression proteins are known to one skilled in
the art. They include, but are not limited to, P58.sup.IPK and BiP
(Yan et al. PNAS 2002, 15920; Van Huizen J. Biol. Chem., 2003,
15558), eIF2, eIF2B, eIF4E, and p97 (Mikami et al Protein Expr
Purif. 2006, 46(2):348-57), K3L and E3L (Davies et al. J Virol.
1993 March; 67(3):1688-92), .gamma..sub.134.5 (He et al. J. Biol.
Chem., 1998, 273:20737), GADD34 (Novoa et al. J Cell Biol, 2001,
1011; Jousse et al. J Cell Biol, 2003, 767), CReP (Jousse et al. J
Cell Biol, 2003, 767; Connor et al. Mol Cell Biol, 2001, 6841), PP1
regulatory subunit (Egloff et al. EMBO J 1997, 1876), Nck-1
(Kembache et al. JBC, 2004 9662; Kembace et al PNAS 2002, 5406),
IMPACT (Pereira JBC, 2005, 28316), eIF2B (Kembache et al. J. Biol.
Chem., 2004 9662).
[0035] An ATP-regeneration system is known in the art and includes
creatine kinase (CK) and creatine phosphate. Additional
supplemental nucleotides required for in vitro transcription and
translation include adenosine triphosphate (ATP), cytosine
triphosphate (CTP), guanidine triphosphate (GTP), and uracil
triphosphate (UTP).
[0036] In any of the disclosed embodiments, the cell-free extract
can be prepared using cultured cells, e.g., 3T3 mouse cells,
Chinese hamster ovary (CHO) cells, etc., known to one skilled in
the art. The cultured cells may be human-derived. The cultured
cells may be grown in media containing heavy isotope-labeled amino
acids.
[0037] The heavy isotope may be an isotope that is different in
abundance than the naturally occurring isotope. The final
concentration of the heavy isotope-labeled amino acid in the in
vitro translation system may be 0.05 mM to 4 mM. The final
concentration of the heavy isotope-labeled amino acid in the
coupled in vitro transcription/translation system may be 0.5 mM to
4 mM. The heavy isotope may be a stable isotope selected from,
e.g., .sup.74Se, .sup.76Se, .sup.77Se, .sup.78Se, .sup.82Se,
.sup.15N, .sup.13O, .sup.18O, and .sup.2H.
[0038] In any embodiment of the inventive method, the incorporation
efficiency of the heavy isotope-labeled amino acid ranged from 10%
to 100%.
[0039] In any embodiment using a coupled in vitro
transcription/translation system, the method may be performed in a
dialysis chamber. This resulted in relatively higher yield of the
at least one heavy isotope-labeled protein from the DNA, compared
to incubation not in a dialysis chamber. The heavy isotope-labeled
amino acids were added to the external dialysate containing
additional limiting reaction components such as salts, nucleotides,
tRNAs, and additional non-heavy isotope-labeled amino acids. In
this method, IVT reactions expressed proteins for longer periods of
time, resulting in higher protein yield.
[0040] Uses of the heavy isotope-labeled protein include, but are
not limited to, the following uses. The heavy isotope-labeled
protein may be purified. The heavy isotope-labeled protein may be
quantitated, either before or after purification. As one example, a
fluorescent protein tag may be part of the expressed protein
sequence, and fluorescence may be determined using standard methods
known in the art to quantitate the expressed protein. As one
example, the heavy isotope-labeled protein may be quantitated by
mass spectroscopy using at least one reference peptide from the
protein or from a protein tag. The heavy isotope-labeled protein
may be quantitated by, e.g., enzyme linked immunosorbent assay
(ELISA), Western blot, and/or antibody-based quantitative assay, as
known in the art. The purified heavy isotope-labeled protein may be
quantitated by a protein assay method, e.g., Bradford assay,
bicinchoninic acid (BCA) assay, and/or Lowry assay, as known in the
art.
[0041] The heavy isotope-labeled protein may be used a standard for
quantitative MS. The heavy isotope-labeled protein may be used for
NMR structural analysis. The heavy isotope-labeled protein may be
used to assess relative recovery of a corresponding native and
heavy isotope-labeled protein during lysis, fractionation,
enrichment, purification, immunoprecipitation, etc. The heavy
isotope-labeled protein may be used to assess relative digestion
efficiency of a native protein and the heavy isotope-labeled
protein. The heavy isotope-labeled protein may be used to assess
relative post-translational modification of a native protein and
the heavy isotope-labeled protein by, e.g., a post-translational
protein modification enzyme, such as a protease, a kinase, a
phosphatase, an acyl transferase, and/or a ligase.
[0042] One embodiment is a kit for producing heavy isotope-labeled
proteins. Kit components of an in vitro translation system include
a mammalian cell-free extract, a plurality of accessory expression
proteins, an ATP regeneration system, a plurality of heavy
isotope-labeled amino acids, and instructions for using the kit to
form an in vitro translation system. Kit components of a coupled in
vitro transcription/translation system include a mammalian
cell-free extract, an RNA polymerase, a plurality of accessory
expression proteins, an ATP regeneration system, a plurality of
heavy isotope-labeled amino acids, and instructions for using the
kit to form an in vitro transcription/translation system.
[0043] One embodiment is a kit for producing heavy isotope-labeled
proteins in a dialysis chamber. Kit components include a mammalian
cell-free extract, an RNA polymerase, a plurality of accessory
expression proteins, an ATP regeneration system, a plurality of
heavy isotope-labeled amino acids, and instructions for using the
kit to form an in vitro transcription/translation system in a
dialysis chamber.
[0044] Methods for quantifying biomolecules standards expressed
using mammalian cell-free extracts. One embodiment is a method
quantifying a protein standard using a protein assay. One
embodiment is quantifying a protein standard using an assay such as
absorbance at 280 nm (A.sub.280), Bradford assay, Lowery assay, BCA
assay, or 660 nm assay. One embodiment is a method quantifying a
protein standard using a fluorescent assay. One embodiment is
quantifying a protein standard that is a fluorescent protein. One
embodiment is quantifying a protein standard that is expressed with
a fluorescent tag. One embodiment is a protein standard where the
fluorescent tag is a fluorescent protein. One embodiment is a
protein standard where the fluorescent tag is GFP. One embodiment
is quantifying a recombinant protein standard using a reference
peptide. One embodiment is using a peptide from the protein
standard sequence as a reference peptide. One embodiment is using a
peptide co-expressed with the protein standard as a reference
peptide.
[0045] Methods for quantifying isotope-labeled protein standards
for use as a relative or absolute standard with mass spectrometry.
One embodiment mixes a stable isotope-labeled protein standard with
a sample containing the protein of interest to be measured. In one
embodiment, a stable isotope-labeled protein standard is not
purified before mixing with samples containing the proteins to be
measured by relative and/or absolute abundance. In one embodiment,
a stable isotope-labeled protein standard is purified before mixing
with samples containing the proteins to be measured by relative
abundance. In one embodiment, a stable isotope-labeled protein
standard is serially diluted to create a standard curve for
absolute quantitation. In one embodiment, a stable isotope-labeled
protein standard is serially diluted before mixing with unlabeled
samples.
[0046] In one embodiment, the disclosed isotope-labeled proteins
produced with an IVT system were purified from the IVT reaction
mixture prior to use as a MS protein standard. In one embodiment,
the disclosed isotope-labeled proteins produced with an IVT system
were unpurified from the IVT reaction mixture prior to their use as
a MS protein standard. In one embodiment, the isotope-labeled
protein was quantitated, e.g., by MS or by measuring fluorescence
of the isotope-labeled protein. In one embodiment, the IVT reaction
mixture or an aliquot of the IVT reaction mixture was "spiked",
i.e., supplemented, with a known quantity of a heavy labeled
peptide, and the "spiked" IVT reaction mixture was then analyzed by
MS to quantitate the isotope-labeled protein based on a comparison
to the heavy labeled peptide. In one embodiment, the "spiked"
peptide had a fluorescent tag and the isotope-labeled protein also
had a fluorescent tag, allowing the absolute quantity of the
IVT-expressed protein to be determined by comparing the fluorescent
intensities of the "spiked" peptide and the expressed protein from
the IVT reaction mixture. In one embodiment, the heavy proteins
were "spiked" in whole. In one embodiment, the heavy peptides were
"spiked" pre-digested by, e.g., exposing the IVT reaction mixture
to a proteolytic enzyme (e.g., trypsin) or a proteolytic agent, to
determine digestion efficiency. In one embodiment, digestion
efficiency ranged from 10% to 100%. By determining the amount of
the expressed isotope-labeled protein in the IVT reaction mixture,
the unpurified expressed isotope-labeled protein in the IVT
reaction mixture was used to "spike" an unknown sample and
therefore, be used as a quantitative internal standard.
[0047] One embodiment is a reagent kit for protein synthesis for
implementing the disclosed method. The kit may include, as known to
one skilled in the art, the following components at concentrations
adequate for protein synthesis, a cell-free extract, accessory
proteins, RNA polymerase, nucleotide mixture, a mixture of ATP,
creatine phosphate, and creatine phosphokinase ("energy" mixture),
salts, RNAse inhibitors, tRNA amino acid mixture, stable
isotope-labeled amino acids, and nuclease-free water, with
instructions for performing the method.
[0048] FIGS. 1A and 1B schematically show use of a human cell
extract in in vitro protein expression (IVT). Heavy isotope-labeled
recombinant protein expression, purification, and mass spectroscopy
(MS) analysis are shown. HeLa cells were grown in a standard
suspension media, then harvested, and a cell-free extract or lysate
was obtained. In FIG. 1A, cells were cultured with normal or SILAC
media, harvested, and lysed to prepare cell lysates. Lysates were
combined with reaction mixture, vector DNA, and stable
isotope-labeled amino acids to express recombinant proteins in an
IVT reaction. The expressed protein was purified, digested into
peptides, and analyzed by LC-MS (FIG. 1B). Percent incorporation of
stable-isotopes was based on ratio of areas under the curve (AUC)
for heavy and light isotopically-labeled peptides.
[0049] Cells were cultured and lysate prepared as follows. HeLa
cells (ATCC CCL-2.2) were grown in suspension using SMEM
supplemented with 10% FBS. For stable-isotope labeling using amino
acids in cell culture (SILAC), HeLa cells were grown in custom SMEM
media supplemented with .sup.13C.sub.6.sup.15N.sub.2 L-Lysine,
.sup.13C.sub.6.sup.15N.sub.4 L-arginine and 10% dialyzed FBS.
Cell-free extracts were prepared by exposing cells to nitrogen
under hyperbaric conditions (300 psi), followed by release from a
PARR non-stirred pressure vessel at a constant rate.
[0050] Isotopically-labeled proteins were expressed in IVT
reactions using a custom amino acid mix supplemented with stable
isotope-labeled amino acids .sup.13C.sub.6.sup.15N.sub.2 L-Lysine
and .sup.13C.sub.6.sup.15N.sub.4 L-arginine. All reactions were
incubated at 30.degree. C. for up to 16 hours. Recombinant HIS-GFP
protein fluorescence was measured using a GFP standard curve with a
Tecan Safire fluorometer and purified using the Pierce HisPur
Cobalt Resin Purification Kit.TM. (Pierce Biotechnologies, Inc.
Rockford Ill.). Recombinant HA-tagged proteins were assessed by
Western blotting using anti-HA antibodies and purified using the HA
Tag IP/Co-IP Kit (Pierce Biotechnologies, Inc.).
[0051] Samples were prepared and subjected to LC-MS as follows.
Purified protein samples were separated by SDS-PAGE and stained
using GelCode.RTM. Blue Stain Reagent (Pierce Biotechnologies,
Inc.). Gel slices containing each protein were destained, reduced
and alkylated, before digestion to peptides. Digestion occurs by
exposure to a proteolytic enzyme (e.g., trypsin) or other
proteolytic agent. In this case, digestion was performed using
trypsin for 4-16 hours. After digestion, the peptides were desalted
using Proxeon Stage C18 micro-column tips (Thermo Fisher
Scientific, Inc.) and reconstituted with 0.1% TFA.
[0052] For peptide analysis, a NanoLC-2D.TM. high-pressure liquid
chromatograph (HPLC) (Eksigent) with a PicoFrit.TM. C18 column 75
.mu.m ID.times.20 cm (New Objective) was used to separate peptides
using a 5%-40% gradient (A: water, 0.1% formic acid; B:
acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min for 40
min. A Thermo Scientific LTQ Orbitrap XL ETD Mass Spectrometer was
used to detect peptides using a top six experiment consisting of
single stage MS followed by acquisition of six MS/MS spectra with
collision induced dissociation (CID) for protein
identification.
[0053] Using the Thermo Scientific Proteome Discoverer.TM. 1.3
software, CID spectra were searched using the SEQUEST.RTM. search
engine against a custom human SWISProt database. Static
modification included carbamidomethyl with methionine oxidation,
Lysine-8 and Arginine-10 used as dynamic modifications. SILAC
ratios were based on the area under the curve (AUC) for each heavy
and light peptide and used to determine stable isotope
incorporation.
[0054] To measure protein abundance using MS, heavy protein
standards are required to accurately quantify the amounts of
unlabeled, endogenous proteins in samples. Ideal protein standards
are identical to their endogenous counterparts. Expression of
recombinant proteins in human cell-free extract systems aids in
proper protein folding and post-translational modifications by,
e.g., one or more of a protease, kinase, phosphatase, methyl
transferase, methylase, acetyl transferase, or acetylase.
[0055] FIGS. 2A-2D show results of isotope-labeled green
fluorescent protein (GFP) expression using human IVT extracts using
two different methods for heavy isotope-labeling. One method used
heavy SILAC-labeled cells to produce a heavy isotope-labeled IVT
extract. The other used normal light IVT extracts supplemented with
heavy isotope-labeled amino acids. Both methods successfully
expressed GFP with stable isotope incorporation >95%.
[0056] FIG. 2A shows expression of GFP in IVT reactions using HeLa
cell lysates derived from cells grown in normal light media that
contained normal 10% standard fetal bovine serum; SILAC light media
which contained 10% dialyzed fetal bovine serum, 0.1 mg/ml light
L-arginine and 0.1 mg/ml L-lysine; or SILAC heavy media that
contained 10% dialyzed fetal bovine serum, 0.1 mg/ml
.sup.13C.sub.6.sup.15N.sub.4 L-arginine and 0.1 mg/ml
.sup.13C.sub.6.sup.15N.sub.2 L-lysine. Protein expression was
determined using GFP fluorescence and purified protein staining
(GelCode Blue). Light 1 and 2 show typical IVT coupled-protein
expression from two different lots of normal lysate. FIG. 2B shows
a representative mass spectrum of heavy/light peptide pair used to
determine SILAC ratios, i.e., heavy peptide peak area compared to
light peptide peak area, and stable-isotope incorporation
efficiency. Efficiency was assessed as the percentage of peptide MS
signal intensity converted from the light to heavy form. FIG. 2C
shows coupled IVT expression of GFP and percent incorporation of
stable isotope amino acids in IVT reactions with increasing
concentrations of stable isotope-labeled lysine and arginine added
to normal, light HeLa protein extracts. FIG. 2D shows coupled IVT
expression of GFP and percent incorporation of stable isotope amino
acids in GFP with increasing time of incubation using normal, light
HeLa protein extracts supplemented with heavy isotope-labeled amino
acids.
[0057] The light lysates had a significantly higher level of
protein expression and lower cost of production (FIG. 2A).
Titration and time course experiments using the light lysate with
heavy amino acids showed that amino acid concentrations >1 mM
and incubation times longer than four hours were necessary for
optimal protein expression and isotope incorporation (FIGS. 2C,
2D).
[0058] FIGS. 3A-3B schematically show a high-yield human coupled in
vitro transcription/translation expression of isotopically-labeled
GFP. This high yield embodiment uses a dialysis chamber containing
supplemental isotopically labeled amino acids to express heavy
isotope-labeled proteins. (FIG. 3A). GFP expression and stable
isotope incorporation efficiency are shown in FIG. 3B. The
high-yield human IVT extracts were supplemented with 0, 0.4 mM, 1.2
mM and 3.6 mM heavy isotope-labeled amino acids in the dialysate.
Heavy labeled-isotope incorporation was 64% with IVT extracts that
were supplemented with 0.4 mM heavy-isotope-labeled amino acids.
Heavy labeled-isotope incorporation was 83% with IVT extracts that
were supplemented with 1.2 mM heavy-isotope-labeled amino acids.
Heavy labeled-isotope incorporation was 95% with IVT extracts that
were supplemented with 3.6 mM heavy-isotope-labeled amino
acids.
[0059] To demonstrate the use of stable isotope-labeled GFP as a MS
quantitation standard, purified heavy protein was spiked into light
GFP samples at different dilutions. FIG. 4 shows linearity of MS
quantification using a heavy isotope-labeled protein standard,
i.e., stable isotopically-labeled GFP as a standard for MS
quantitation of a non-heavy-isotope labeled or light GFP standard
dilution. A titration curve of purified heavy isotope-labeled GFP
and light GFP, using a fixed amount of stable isotope-labeled GFP
(10 fmol), was mixed with unlabeled GFP in ratios from 27:1 to
1:27. Experimental values were fitted to a linear regression line
using theoretical expected values. There was a high correlation
coefficient (R.sup.2 >0.98) for both arginine and lysine
containing peptides over a broad range of peptide concentration
ratios.
[0060] Six additional recombinant proteins were expressed and
purified using HA-tag affinity purification to further demonstrate
the inventive method for production of human-based proteins. The
results are shown in FIGS. 5A-D. FIGS. 5A and 5B show Western blot
results of stable isotope incorporation in six different proteins,
GFP, BAD, CyclinD1, p53, RB, and GAPDH, expressed using human IVT
extracts that were supplemented with heavy amino acids. In FIG. 5A,
* indicates an anti-GST cross-reacting band in the lysate. FIG. 5C
is a representative MS spectrum of a heavy labeled GAPDH peptide.
FIG. 5D is a representative MS spectrum of a heavy labeled cyclin
D1 peptide.
[0061] Although all proteins were expressed, as indicated by a
Western blot, only four out of the six proteins were recovered
after purification and sample preparation. Of the proteins
identified by MS, all had stable isotope percent incorporations
near or exceeding 95%. These results substantiated the
applicability of the disclosed IVT system to create heavy protein
standards for MS quantitation.
[0062] FIGS. 6A-B show expression of a heavy isotope BAD protein
and MS analysis. FIG. 6A shows a Coomassie-stained SDS-PAGE gel of
recombinant light (left side) and heavy isotope-labeled (right
side) BAD-GST-HA-6.times.HIS purified from HeLa IVT lysates (L),
using glutathione resin (E1) and cobalt resin (E2) tandem affinity,
with flow throughs (FT) from each column indicated. Sequence
coverage and representative MS spectra of stable-isotope-labeled
BAD are shown in FIG. 6B; there was >95% isotope
incorporated.
[0063] The inventive method and system will be further appreciated
in view of the following non-limiting examples.
EXAMPLE 1
Heavy Protein Production Using a Human IVT System Derived from
SILAC-Labeled Cells
[0064] A SILAC-labeled HeLa cell lysate was used because this
lysate had the highest probability of producing an
isotopically-labeled protein. The endogenous amino acids L-lysine
or L-arginine and tRNAs linked to these amino acids would be
isotopically labeled after 4-5 cell doublings of cells grown in
media containing only .sup.13C.sub.6.sup.15N.sub.2 L-lysine-2HCl
and/or .sup.13C.sub.6.sup.15N.sub.4 L-arginine-HCl. All proteins in
the HeLa cells were >98% labeled using SILAC, determined by MS
analysis of lysate protein tryptic peptides. A lysate was prepared
using a standard method to produce a cell-free extract amenable to
coupled-IVT which is capable for both transcription and translation
in a single extract. Test expression of a model protein HIS-tagged
GFP from vector DNA confirmed that the heavy lysate was capable of
protein expression; protein expression yields were about five-fold
less than control unlabeled lysates grown in normal media. This
result was reproducible and appeared to be a consequence of poorer
cell health before lysate production, possibly linked to the use of
dialyzed FBS required for the SILAC method. Despite the low yield
of HIS-GFP expression using heavy lysates, the average isotope
incorporation for GFP protein based on MS of tryptic peptides was
>95% when protein was expressed using extracts supplemented with
additional heavy amino acids and exogenous tRNAs were omitted.
[0065] This demonstrated production of a heavy protein using a
mammalian IVT system. The cost of this method was high and somewhat
wasteful because the entire lysate was heavy labeled when only
labeling of the expressed protein was required for production of a
protein standard. In both control experiments using heavy lysates
derived from SILAC cells supplemented with light amino acids, and
normal light lysates supplemented with heavy amino acids in
reaction mixtures, there was about 30-40% isotope incorporation for
both controls after MS analysis of HIS-GFP. Omission of exogenous
tRNAs increased isotope incorporation by an additional 10%, to a
final average incorporation of 50%. These high levels of
incorporation were not expected because traditional eukaryotic IVT
systems, e.g., wheat germ or rabbit reticulocyte, typically have
<5% isotopically-labeled amino acid incorporation unless they
are modified.
EXAMPLE 2
Heavy Protein Production Using a Human IVT System Supplemented with
Heavy Amino Acids
[0066] An efficient and effective way to produce
isotopically-labeled proteins was to add heavy amino acids into
normal light IVT reaction mixtures. The amount of heavy amino
acids, .sup.13C.sub.6.sup.15N.sub.2 L-lysine-2HCl and/or
.sup.13C.sub.6.sup.15N.sub.4 L-arginine-HCl, added to light lysates
were titrated from 0 mM, 2 mM, 5 mM, 10 mM, 50 mM, or 100 mM,
keeping the remaining amino acid concentrations constant at 2 mM.
The isotope incorporation efficiency of expressed HIS-GFP was
>95% at heavy amino acid at concentrations >50 mM. In this
system, adding exogenous tRNA did not result in decreased isotope
incorporation. These results were not expected because the
concentration of the amino acid supplement was 2 mM for all amino
acids and was believed not to be limiting, i.e., was in excess, for
production of proteins in the coupled-IVT system. The amino acid
titration experiment was repeated with another protein, HA-tagged
BAD, with the same results as for HIS-GFP.
[0067] Using the disclosed methods, additional proteins RB, cyclin
D1, p53, and GAPDH were expressed and isotope incorporation
efficiencies were >95%, as shown in FIG. 5. Heavy protein
expression levels observed with traditional methods were about 20%
higher than traditional light coupled lysates and were about 4-5
times higher than using a SILAC-labeled lysate. A HIS-GFP
expression time course indicated that the inventive heavy amino
acid supplement mix may be the source of the higher expression.
Different light amino acid concentration mixtures without lysine
and arginine provide maximal expression and stable isotope
incorporation for heavy amino acids
[0068] The disclosed method of heavy protein production was
significantly less expensive than using a heavy labeled lysate.
[0069] During purification of over-expressed heavy HA-BAD, two
additional proteins co-purified. Using MS, these proteins were
determined to be 14-3-3 proteins that are known to form a
heterodimeric complex with phosphorylated BAD. There are numerous
sites of modification for phosphorylated BAD peptides, including
sites known to be required for 14-3-3 binding. Thus, the function
of the proteins expressed using the disclosed heavy IVT system were
established; the heavy proteins are being modified and integrated
into protein complexes, evidencing both post-translational
modification (phosphorylation) and proper folding.
[0070] The method is used for both coupled IVT and linked IVT,
starting with either DNA or mRNA. The method, in one embodiment,
used a dialysis membrane chamber to create an IVT reaction vessel,
which was surrounded by a large volume of reaction mixture
components that were limited (include amino acids, "energy" mix,
and nucleotides). This allowed higher expression of proteins
without diluting the lysates, which could limit expression. The
expressed heavy proteins are used as a "spike" in standards to
measure changes in endogenous proteins by relative MS
quantitation.
[0071] Five different stable isotope-labeled proteins were produced
using a modified non-SILAC human IVT system. The isotope
incorporation efficiency was >95% for IVT reactions containing
stable-isotope amino acids at concentrations .gtoreq.50 mM and
incubated for longer than four hours. Spike-in experiments using
heavy-labeled protein standards demonstrated MS relative protein
quantification linearity.
EXAMPLE 3
Heavy Isotope-Labeled Protein as an Internal Standard for
Immunocapture Efficiency or Depletion Specificity
[0072] Efficiency of immune capture of proteins depends upon the
affinity, epitope structure, associated factors, specificity,
background, competitors, and other environmental factors (e.g.
salts, lipids, etc.). Using the disclosed methods, a stable
isotope-labeled full length parathyroid hormone (PTH, 1-115 AA) was
expressed and characterized as a C-terminal 6.times.HIS fusion
protein to quantitatively assess sample preparation steps for MS
immunoassays. Light and heavy versions of PTH were expressed at
high levels (>50 .mu.g/mL) and were highly purified using a
cobalt immobilized metal affinity column (IMAC). Heavy PTH isotope
incorporation efficiency was assessed using high resolution mass
spectrometry and measured to be 96.2% heavy protein labeled based
on the average of identified PTH peptides. Light and heavy PTH were
combined to demonstrate linearity of quantitation. Heavy PTH was
spiked into a human plasma sample containing endogenous processed
PTH and the endogenous and heavy PTH were successfully captured
using anti-PTH immune capture columns and analyzed using a
previously described targeted SRM assay (Lopez et al. (2010)
Selected reaction monitoring-mass spectrometric immunoassay
responsive to parathyroid hormone and related variants. Clin Chem.
56(2):281-90). In this example, the spiked heavy PTH was used to
assess PTH capture efficiency and to normalize results for capture
efficiency across multiple samples. In a related example, abundant
proteins, including albumin and immunoglobulins, are removed from
serum or plasma before or after addition of the heavy
isotope-labeled protein standard as an internal standard for
depletion specificity.
EXAMPLE 4
Heavy Protein as an Internal Standard for In Vivo Protein
Processing and Sample Preparation Digestion Efficiency
[0073] Mass spectrometric analysis of proteins is typically
performed after enzymatic digestion or chemical cleavage of the
protein into its constituent peptides. Proteins may also be
proteolytically processed in their native environment, and these
protein variants may be physiologically relevant and/or have
diagnostic utility. For example, assays for monitoring PTH and PTH
variants are important for the accurate diagnosis of endocrine and
osteological diseases. The heterogeneity of PTH has traditionally
been an impediment to developing assays that distinguish
full-length PTH (PTH1-84) from N-terminally truncated PTH (PTH 7-84
and others). Because intact and truncated forms of PTH vary in
biological activity, assays that can accurately quantify the ratio
of intact hormone to its fragments are needed to accurately
determine the amount of biologically active PTH. To date, most
immunoassays used to monitor PTH levels are based on traditional
sandwich ELISA methods that cannot accurately discriminate intact
PTH from truncated PTH. These methods typically employ primary
antibodies to the N-terminus of the hormone, thereby preventing
quantification of any fragments.
[0074] Using the disclosed methods, a stable isotope-labeled full
length PTH (1-115 AA) was expressed and characterized as a
C-terminal 6.times.HIS fusion protein for use as an internal
standard for assessing and quantifying in vivo proteolytic
processing of PTH in serum or plasma. After depletion of abundant
proteins and/or immune capture of the endogenous and heavy PTH,
samples were spiked with unique heavy isoforms of several PTH
peptides and enzymatically digested with trypsin. To monitor the
numerous isoforms of PTH, PTH and PTH variants were quantified
using selected reaction monitoring (SRM) of the depleted and/or
immune-enriched samples, using the spiked heavy peptides as
quantitative standards. The yield of peptides from heavy PTH
digestion is used to assess digestion efficiency and to normalize
digestion efficiency across multiple samples.
EXAMPLE 5
Heavy Protein Production for NMR Structural Analysis
[0075] Methods to make isotope-labeled protein for NMR application
are disclosed. NMR typically required 0.1 mg-1 mg of protein, with
all carbons and/or nitrogens isotopically labeled for structural
analysis. This requires supplementing the lysate with all amino
acids as heavy-isotope labeled amino acids. For this, human
cell-free extractions are derived from cells grown using heavy
carbon or nitrogen-containing media. IVT extracts are supplemented
with reaction components using a mix of amino acids and/or charged
tRNAs with all amino acids labeled with .sup.15N or .sup.13C.
Alternatively, proteins are expressed using normal light human
cell-free extracts supplemented with heavy amino acids. In either
method, protein isotope incorporation efficiency is verified by MS
before NMR analysis. Heavy proteins need to be properly folded and
purified for NMR analysis.
[0076] All references are specifically incorporated by reference
herein in their entirety.
[0077] Applicants incorporate by reference the material contained
in the accompanying computer readable Sequence Listing identified
as Sequence_Listing_ST25.txt, having a file creation date of Jul.
25, 2012 4:22 P.M. and file size of 2.86 KB.
[0078] The embodiments shown and described in the specification are
only specific embodiments of inventors who are skilled in the art
and are not limiting in any way. Therefore, various changes,
modifications, or alterations to those embodiments may be made
without departing from the spirit of the invention in the scope of
the following claims.
Sequence CWU 1
1
5120PRTArtificial SequenceSynthetic Construct 1Val Glu Glu Asp His
Ser Asn Thr Glu Leu Gly Ile Val Glu Tyr Gln 1 5 10 15 His Ala Phe
Lys 20 210PRTArtificial SequenceSynthetic Construct 2Ala Gly Ala
His Leu Gln Gly Gly Ala Lys 1 5 10 311PRTArtificial
SequenceSynthetic Construct 3Ala Tyr Pro Asp Ala Asn Leu Leu Asn
Asp Arg 1 5 10 422PRTArtificial SequenceSynthetic Construct 4His
Ser Ser Tyr Pro Ala Gly Thr Glu Asp Asp Glu Gly Met Gly Glu 1 5 10
15 Glu Pro Ser Pro Phe Arg 20 5168PRTArtificial SequenceSynthetic
Construct 5Met Phe Gln Ile Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp
Ser Ser 1 5 10 15 Ser Ala Glu Arg Gly Leu Gly Pro Ser Pro Ala Gly
Asp Gly Pro Ser 20 25 30 Gly Ser Gly Lys His His Arg Gln Ala Pro
Gly Leu Leu Trp Asp Ala 35 40 45 Ser His Gln Gln Glu Gln Pro Thr
Ser Ser Ser His His Gly Gly Ala 50 55 60 Gly Ala Val Glu Ile Arg
Ser Arg His Ser Ser Tyr Pro Ala Gly Thr 65 70 75 80 Glu Asp Asp Glu
Gly Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg 85 90 95 Ser Arg
Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg 100 105 110
Glu Leu Arg Arg Met Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly 115
120 125 Leu Pro Arg Pro Lys Ser Ala Gly Thr Ala Thr Gln Met Arg Gln
Ser 130 135 140 Ser Ser Trp Thr Arg Val Phe Gln Ser Trp Trp Asp Arg
Asn Leu Gly 145 150 155 160 Arg Gly Ser Ser Ala Pro Ser Gln 165
* * * * *