Page 1 of 42 Articles in PresS. J Appl Physiol (January 10, 2008). doi:10.1152/japplphysiol.00976.2007
Page 1
Determination of Protein Synthesis in vivo Using Labeling from
Deuterated Water and Analysis of MALDI-TOF Spectrum
Gary Guishan Xiaoa, Meena Garga, Shu Lima, Derek Wonga,
Vay Liang Gob, Wai-Nang Paul Leea
a
Department of Pediatrics, Division of Endocrinology, Los Angeles Biomedical Research
Institute at Harbor-UCLA Medical Center and b Department of Medicine, Division of
Running head: Determination of Protein Synthesis Using MIDA
Address for reprint requests and other correspondence:
Wai-Nang Paul Lee
Los Angeles Biomedical Research Institute at Harbor-UCLA
1124 West Carson Street
Torrance, CA 90502
Tel: 310-222-6729, Fax: 310-222-3887
e-mail: [email protected]
Copyright © 2008 by the American Physiological Society.
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Gastroenterology, David Geffen School of Medicine, Los Angeles, California
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ABSTRACT –
This paper describes a method of determining protein synthesis and turnover using in
vivo labeling of protein with deuterated water and analysis of MALDI-TOF spectrum.
Protein synthesis is calculated using mass isotopomer distribution analysis instead of
precursor to product amino acid enrichment ratio. During protein synthesis, the
incorporation of deuterium from water changes the mass isotopomer distribution (isotope
envelop) according to the number of deuterium (0, 1, 2, 3, etc.) incorporated, and the
distribution. A mathematical algorithm by which the distribution of deuterium
isotopomers can be extracted from the observed MALDI-TOF spectrum is presented.
Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation
of their distribution provides a method for the quantitation of newly synthesized proteins.
The combined use of post-source decay sequence identification and mass isotopomer
distribution analysis makes the use of in vivo labeling with deuterated water a precise
method to determine specific protein synthesis.
Keywords: MIDA, isotopomer, spectral analysis, peptide sequence identification
Abbreviations: MALDI-TOF, matrix-assisted laser desorption time-of-flight mass
spectrometer; PSD, post-source-decay
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distribution of the protein with 0, 1, 2, 3, … atoms of deuterium follows a binomial
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INTRODUCTION
The concentration of a protein in a cell depends on the rate of its synthesis and
degradation. Thus, the rate of protein turnover, the time required to synthesize a certain
protein and maintain its concentration in a cell, is a sensitive indicator of cell physiology
or its phenotype. Protein turnover is usually expressed as the rate of protein synthesis (in
mole/unit time or weight/unit time) or its half-life, which is the time required to achieve
half of the maximum concentration of the protein. The determination of protein synthesis
radioactive tracers were used for the determination of protein synthesis and turnover.
With availability of sensitive and precise mass spectrometers, the use of radio-isotopes
has been replaced by application of stable isotope tracers (18). The basic principle of
protein turnover measurement using tracers relies on the measurement of the specific
activity of the “precursor” (or the labeling agent, which can be deuterium in deuterated
water, or specifically labeled amino acids such as [1-13C]-leucine, [5,5,5-2H3]-leucine)
and the determination of the specific activity (SA) or enrichment (E) of the labeling agent
(precursor) in the protein. The newly synthesized fraction (FNP) is provided by the
formula:
FNP = [SA or E of the precursor in protein]/ [SA or E of the precursor] ------ Equation 1
Protein synthesis rate (PSR) is calculated by dividing the quantity of new protein by the
time interval for the change using the equation:
PSR = [protein concentration] × (FNP) / (unit time) ---------------------------Equation 2
Examples of such applications for the determination of protein synthesis and turnover
using tracers have been reviewed by Wolfe RR (21).
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and turnover has been of great interest to biologists for more than 2 decades. Previously
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Recently, Previs and Hellerstein separately introduced a novel method for the
determination of protein synthesis using in vivo labeling with deuterated water (D2O) or
heavy water (16, 3). When cells or animals are given deuterium water, it is possible to
maintain a constant high level of enrichment in water (0.5-2 %). Plasma alanine rapidly
incorporates the deuterium from water changing its CH3- group to CD3- thus increasing
its molecular weight depending on the deuterium enrichment in water (21). The
enrichment in alanine can be determined by gas chromatography/mass spectrometry
as albumin is first isolated and hydrolyzed, and the enrichment in alanine isolated from
the protein is similarly determined by GC/MS. FNP can be calculated using the equation
1. The method of Hellerstein (3) differs from the method of Previs (16) in that a different
experimental approach was used to determine amino acid enrichment using mass
isotopomer distribution analysis (MIDA). It should be noted that these methods are
variations of the basic precursor/product enrichment ratio method. They all require 1)
determination of precursor SA or E, 2) isolation of specific protein of interest, 3)
hydrolysis of the protein to separate the amino acids, 4) determining the enrichment in
the specific amino acid of interest, and 5) application of the equations 1 and 2. A major
disadvantage of this approach is that the accuracy of the calculated protein synthesis
depends on the purity of the protein isolated.
We describe here a novel approach for the determination of protein synthesis and
turnover using 2H labeling and protein mass spectrum. It is known that metabolic
systems in cells of organisms are capable of synthesizing non-essential amino acids. In
the process of amino acid synthesis, carbon, nitrogen and hydrogen atoms are
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(GC/MS) after derivatization. To determine protein synthesis, the specific protein such
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incorporated from their precursor substrates either directly or through exchanges
(transamination). If these precursor substrates are enriched with one or more of these
isotopes, the resultant amino acids are heavier than the “natural” amino acids. Protein
molecules that are synthesized after the introduction of the heavy isotopes will contain
the heavier amino acids resulting in a mass shift in the corresponding spectrum (Figure
Fig. 1
1). The intensities of the mass peaks corresponding to the unlabeled (light) and the
labeled (heavy) can be used for calculation of the newly synthesized fraction. The
all relevant mass peaks of the peptide spectrum instead of the intensities of unique mass
peaks. The mass spectral peaks of the heavier protein are resolved from the lighter
(natural) protein by a mathematical algorithm. The capability of resolving overlapping
spectra makes it possible to label proteins with lower enrichment of stable isotopes. The
need for highly enriched precursor substrates is obviated (22).
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method described herein differs from previous methods in that it utilizes the intensities of
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MATHEMATICAL ALGORITHM
THE BINOMIAL DISTRIBUTION MODEL OF MASS ISOTOPOMERS IN PEPTIDE
In order to be able to separate the “light” spectrum from that of the “heavy” or
labeled spectrum, one has to fully understand the theory that governs the mass
isotopomer distribution of a large molecule such as a peptide and how the incorporation
of heavy isotope subsequently modifies such a distribution. Mass isotopomer distribution
in a peptide spectrum is mostly generated from the presence of 13C at its natural
distribution of isotopomers in the “isotope envelop” can be modeled by a binomial
distribution with parameters N and p. In the case of 13C distribution in peptide, N is the
number of carbon atoms and p is the natural enrichment of 13C (Footnote 1). Assuming
Footnote 1
the 13C natural enrichment of 1.1%, the number of carbon atoms can be estimated from
Footnote 2
the observed distribution using the relationship of consecutive mass ratio (Footnote 2)
M1/M0 = N×p/q, where M0 is the molar fraction of monoisotopic peak and M1 is the
molar fraction of peptide with one 13C (11). The use of this model to simulate mass
isotopomer distribution in the “isotope envelop” is shown in the following example of a
peptide (m/z 1393) from AMP deaminase in an albumin fraction after tryptic digest
Fig. 2
(Figure 2). The intensities of the individual mass peaks were first expressed as a (molar)
fraction of the all the isotopomer species. The theoretical distribution was calculated
Footnote 3
based on N=69 (Footnote 3) and p=0.011. The goodness of fit can be seen by inspection
of Figure 2 and tested by multiple linear regression analysis which showed a coefficient
of 0.96 and a R-squared of 0.99.
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abundance (see below for a more general discussion of isotopes in protein). The
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THE CONCATENATION FUNCTION ( ) OF MASS ISOTOPOMERS
In a molecule consisting of carbon, nitrogen, oxygen and hydrogen, the molecular
weight as well as the distribution of isotopomers can be predicted from the molecular
weights of the elements as well as the frequency of finding their corresponding heavy
isotopes. (19, 24) In general, the isotopomer distribution can be seen as a concatenation
of groups of isotopomers from the same elements –
[carbon isotopomers]
[hydrogen isotopomers]
[sulfur isotopomers]
For the purpose of this paper, we simplify the experimental model to the specific
condition involving deuterium:
[carbon isotopomers] [deuterium isotopomers]
is the concatenation operation which generates the mass isotopomers of the whole
Fig. 3
molecules from the isotopomers of its component parts (Figure 3) (11). For example, if
mass isotopomer distribution in carbon is given by [a0m0, a1m1, a2m2,…] and the
isotopomers from deuterium is [b0m0, b1m1, b2m2,…], the isotopomers of the peptide
that contains both components is given by [a0b0m0, (a0b1+a1b0)m1, (a1b1+a2b0+b2a0)m2,
…]. The sum of the coefficients for each component is equal to 1 ( ai = 1; and bi = 1).
As illustrated by the example, the coefficient of the combined peptide is given by the sum
of the products with subscript indices equal to the mass isotopomer number. In this case,
the coefficient of m0 is given by a0b0 and m1 given by a0b1+b0a1. In fact, the binomial
distribution is mathematically a special case of the concatenation function.
Fig. 4
Figure 4 shows a hypothetical example of deuterium incorporation from
deuterated water into a peptide. The original peptide is represented by carbon
isotopomers. When such a peptide is synthesized in the presence of deuterated water,
each peptide molecule may incorporate 1, 2 or 3 deuterium depending on the enrichment
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[nitrogen isotopomers]
[oxygen isotopomers]
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in water. The final distribution of the heavy peptide is the result of concatenation of
carbon-isotopomers and the deuterium isotopomers. In this example the distribution of
carbon isotopomers is given by the binomial distribution with N(c)=100 and p(c)=0.011
and the deuterium isotopomer distribution is given by N(d) = 20 and p(d) = 0.04. These
distributions are shown in panels A and B. Using the concatenation operation on these
two distributions, we arrived at the distribution of the combined distribution. (Appendix)
Panel C shows the isotopomer distribution of the resultant peptide as observed in the
mass spectrum of the peptide. Panel D show the changes in isotopomer distribution
(mass spectrum) of the same peptide under 2% and 4% deuterium enrichment conditions.
spectrum and the “unlabeled” spectrum, and converts it to a deuterium isotopomer
spectrum using regression analysis. It can be shown that the
operation on a peptide
without deuterium label (pre-existing protein) returns the result m0 = 1 or unlabeled
(Footnote 4). Since m0 of the deuterium spectrum can come from the newly synthesized
Footnote 4
peptide or the pre-existing peptide, the deuterium isotopomers of the deuterated protein is
diluted by m0 contributed by the unlabeled protein. The ratio between the observed and
the theoretical molar fractions of m1, m2 etc from the deuterium isotopomers of the
newly synthesized peptide give the fraction of newly synthesized peptide (116). (Figure
5)
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The mathematical algorithm of the inverse operation ( ) takes the observed
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MATERIALS AND METHODS
Animal protocol: The animal study was carried out according to a protocol approved by
Animal Research Committee at UCLA. Sprague Dawley rats (250 gm) were treated with
a priming dose of D2O equal to 6% body water and maintained on drinking water
enriched with 4% deuterium as previously described (10). Serum was obtained before
and on the 8th day of deuterated water treatment. Serum albumin was purified from these
samples using AffiGel-Blue
affinity column. The albumin fraction was eluted with
was determined by its optical density (OD280). Pretreatment sample and post-treatment
samples were mixed in the proper proportions of protein concentrations to simulate 10,
25, 50 and 75% synthesis. The proteins in these samples were precipitated using 10%
trichloroacetic acid. The desalted sample was concentrated before trypsin digestion and
analysis by mass spectrometer.
Acquisition of mass spectra by MALDI-TOF: Trypsin digestion was performed by
following the standard protocol (17, 23) with minor modification. The purified albumin
fractions were prepared in 50 mM NH4HCO3. A 50 mL aliquot was removed and mixed
with 50 mL of ACN to improve proteolysis (17). Trypsin (Trypsin Gold, Promega, CA)
was added to a final protease-to-protein ratio of 1:80, and the digestion mixture was
incubated at 37 °C for 24 h. The digested peptides were lyophilized and re-suspended in
50% acetonitril/0.3% trifluoric acid (TFA), and purified by using C18 zip tips (Millipore,
CA) prior to MALDI TOF profiling and post-source decay sequence identification using
a Voyager DE (Voyager-DE workstation, Applied Biosystems, Forster, CA) (23).
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1.4M NaCl in 20 mM Tris buffer (pH 7.1). Protein concentration in the albumin fraction
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The purified peptides using C18 zip tip was directly eluted to MALDI plate with 2 µl of
10 mg/ml a-cyano-4-hydroxysinomic acid (CHCA), and allowed to dry. The molecular
weight of each sample was measured on a MALDI mass spectrometer with positive mode
using Voyager DE reflector mode MALDI-Time-of-Flight (TOF). The instrument was
calibrated with insulin standard (ProteoMass Insulin MALDI-MS standard [5729.6089]
and ACTH standard (ProteoMass ACTH fragment 18-39 MALDI-MS standard
[2464.1989]) (Sigma, St. Louis, MO). The mean molecular mass was calculated from
plate, high laser power was applied (2175 vs. 1970 U for the standards). The instrument
operated in positive ion delayed extraction mode with a low-mass gate of 500 Da. The
accelerating voltage was 25,000 V (non digested insulin and glucagons) or 20,000V
(trypsin digested insulin and glucagons), the grid voltage was set to 65%, the delayed
time is 150 nsec (trypsin digested insulin and glucagons) or 285 nsec (non digested
insulin and glucagons). Each spectrum was the average of approximately 200 scans, and
calibrated using external ACTH and insulin calibration files generated during the
experiment.
Identification of tryptic digest by post source decay (PSD): Tryptic digest of rat albumin
was identified using post-source decay (PSD) matrix-assisted laser desorption/ionization
(MALDI) time-of-flight mass spectrometry (6, 7). PSD fragment ion spectra were
acquired for several peptides and peptide derivatives after isolation of the appropriate
precursor ion by using timed ion selection. Fragment ions were refocused onto the final
detector by stepping the voltage applied to the reflectron in the following ratios: 1.0000
(precursor ion segment), 0.9126, 0.6049, 0.4125, 0.2738, 0.1975, and 0.1213 (fragment
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multiple independent measurements. For the extraction of the protein from the MALDI
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segments). The individual segments were stitched together by using software provided by
Applied Biosystems. The MS data from both tandem mass spectra from the LC-MS/MS
experiments and the MALDI-MS PSD mass spectra were searched against a subset of
rodent proteins in the MSDB protein sequence data base, using the Mascot search
program (Matrix Science, London, United Kingdom) (www.matrixscience.com). Positive
protein identification was based on standard Mascot criteria for statistical analysis of the
MALDI peptide fingerprint mass spectra and the LC-MS/MS data. A -10log(P) score,
regarded as significant (5).
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where P is the probability that the observed match is a random event, of >72 was
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RESULTS
Serum was collected from a Sprague Dawley rat after eight days of deuterated water
treatment. Albumin fraction was purified by AffiGelBlue
affinity column. After
tryptic digest, the sample was analyzed by MALDI-TOF for peptides in the range of
1000-2000 daltons. Five peptides were identified using MALDI-TOF and post-source
decay analysis. Their sequences were matched to rat serum albumin, AMP deaminase and
Table 1
alpha 2u-globulin (Table 1). Despite our attempt to purify albumin, the MALDI-TOF
corresponding normalized spectra of these peaks from the sample before deuterated water
Table 2
treatment (unlabeled spectra) are shown in Table 2. Average mass was calculated using
method of Blom (2).
Table 3
Table 3 shows the normalized spectra of peptides isolated from serum after 8 days of
deuterated water treatment. The incorporation of deuterium in these peptides results in
small shifts in the average mass. The magnitude of the shift due to deuterium
incorporation depends on the enrichment in water, the number of non-essential amino
acids and the fraction of new protein. The fraction of new protein was calculated using
the inverse concatenation operation. Examples of such a calculation are shown in the
Appendix. Since the operation utilizes information from all spectral peaks, an estimate of
the accuracy (standard deviation) can be obtained from a single spectrum in m/z 1681.
From the magnitude of mass shift and the calculated new fraction, the number of possible
Footnote 5
deuterium substitution (N(d)) can be estimated (Footnote 5). It is to be expected that
peptides of the same protein have the same synthesis rate and new fraction, which is
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spectrum indicated contamination of albumin by other serum proteins. The
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Table 2
shown in the albumin peptides in Table 2. Peptides from other proteins may have
different synthesis rates and have different new fractions.
The fraction of new protein can also be determined from the peptide spectrum when the
spectra of the “labeled” and “unlabeled” peptides are known. In the example of AMP
deaminase, we found that the protein was almost completely labeled (100% new
synthesis) by day 8 day of deuterium water treatment. Protein fractions containing
after deuterium labeling were mixed in to simulate 10, 25, 50 and 75 % synthesis. The
different mixtures were subject to trypsin digest and MALDI-TOF analysis. The
Table 4
observed spectra are shown in Table 4. Using multiple linear regression analysis, we
determined the coefficients for “labeled” and “unlabeled” peptides that would give the
observed distributions. The coefficient representing percent synthesis and its standard
deviation were provided by the regression analysis (Table 4). Linear response of
isotopomer distribution due to mixing of “labeled” and “unlabeled” peptides is shown in
Fig. 6
Figure 6. As expected, the standard deviation for each measurement increases as percent
synthesis decreases. Therefore, protein synthesis and protein concentration can be
determined from proteins that are maximally enriched (i.e. 100% newly synthesized).
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albumin and AMP deaminase from sera obtained before deuterium labeling and on day 8
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DISCUSSION
Mass isotopomer distribution analysis has been used to determine protein synthesis and
turnover. Specific labeled amino acids of high enrichment are required.
Papageorgopoulos et al. (15) attempted to determine protein synthesis in vivo using mass
isotopomer distribution analysis (MIDA) based on their method for the determination of
synthesis of biopolymers (9, 14). As a demonstration, rats were infused with [5,5,52
H3]leucine (99% enriched) via the jugular catheter for 24 h at a rate of ~ 50 mg/kg/h
Trypsin digest of the protein was analyzed using an electrospray ionization/magnetic
sector mass spectrometer. Mass isotopomers containing leucine isotope in peptides rich
in leucine was determined. Incorporation of [5,5,5-2H3]leucine would result in mass shift
of +3, +6, etc. depending on the number of leucine in the peptide and the enrichment of
intramyocyte leucine. However due to the low protein turnover rate, insufficient amount
of the +3 or +6 isotopomers were detected. Even though MIDA method is theoretically
possible in such application, it is not feasibility to maintain a high infusion of the labeled
precursor to determine synthesis of proteins that turnover slowly. Because of the high
enrichment of specific labeled leucine required, the MIDA method as described by
Papageorgopoulos et al. (15) for in vivo study is not a practical one. Another method
using mass spectrum for the determination of protein synthesis is that of Cargile et al. (8).
The method introduces 13C carbon into amino acids from [U13C6]-glucose (final
enrichment >50%) via amino acid synthesis in bacteria. In organisms that can synthesize
essential and non-essential amino acids from glucose and nitrogen, [U13C6]-glucose
effectively replaces 12C with 13C in protein creating a heavy protein which can be
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using a minipump. Muscle was harvested and creatine kinase (CK) was isolated.
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separated by mass spectrometry. By quantitating the intensity of the labeled and the
unlabeled peaks, a synthesis/degradation ratio was calculated to represent relative protein
turnover dynamics. Such a method is of limited use in higher organisms which can only
synthesize their non-essential amino acids from glucose. Furthermore such an approach
is expensive because of large amount of highly enriched glucose required.
There are major differences distinguishing the described mass isotopomer
analysis from previous in vivo methods. The present method differs from those of Previs
amino acid in protein is not required and 2) fraction of new protein is determined from
the mass spectrum of the protein or its fragments after enzyme digestion. A major
weakness of previous published deuterated water methods is their dependence on the use
of equation 1. It is well known that the isotope enrichment of plasma amino acids usually
do not represent the enrichment in the intracellular amino acids which are the immediate
precursors for protein synthesis. As shown in Table 1, it is difficult to be certain of the
purity of the protein isolated before hydrolysis. Any contamination by other proteins in
the purification potentially introduces error in the determination of amino acid
enrichment in the isolated protein. The described method permits the separate
identification of peptide by post-source decay sequence identification. Thus, protein
synthesis determined from a specific peptide is truly the synthesis of the protein without
ambiguity.
With the advent of high resolution mass spectrometer capable of resolving
molecules with molecular weights (m/z) >2000 daltons, many isotope labeling
approaches have been devised to quantify proteins, determine relative protein expression
and determining protein synthesis. The use of stable isotopes in the quantitation of
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et al. (16) and Busch et al. (3) in that 1) determination of isotope enrichment in specific
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proteins has been reviewed (1, 12, 20). These methods are used in the determination of
(i) relative quantities (expression) (13), (ii) protein identification, and (iii) protein
turnover (synthesis) (4, 9, 14). Generally, these methods utilize specific labeled
precursor of high enrichment and quantitative analysis is carried out by mass peak ratios.
Determination of low levels of deuterium incorporation into peptides from deuterated
water was recently described by Wang et al. (19) by determining excess molar ratio at m1
(peptide with one deuterium incorporated). However, such an approach using excess
mass calculation is similar to the excess M1 calculation of Hellerstein (8), and cannot be
used to determine protein synthesis without additional information of the isotopomer
distribution of the new peptide. From the examples presented above, it is clear that
enrichment from deuterated water when the mass isotopomer distribution of the newly
synthesized peptide is sufficiently different from the distribution in the peptide before
labeling. Since the use of deuterium enriched water for human studies is limited to
enrichment below 0.5%, the present labeling with deuterated water is probably not
suitable for clinical studies, and the use of 13C or 15N labeling for the determination of
protein synthesis in the clinical settings remains to be investigated. The current method
of using in vitro or in vivo labeling of protein and mass isotopomer analysis after partial
purification is certainly a useful approach in the studies of protein turnover and protein
expression in proteomics.
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protein synthesis can be determined from isotope incorporation at low level of
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FOOTNOTES
Footnote 1: There are two parameters in a binomial distribution: the number of trials (N)
and the probability of success (p). In the case of 13C distribution in peptide, N is the
number of carbon atoms and p is the natural enrichment. The natural abundance of 15N
and 18O affects the accuracy of determining the number of carbon atoms in the peptide
but not the overall isotopomer distribution in the “isotope envelop”.
Footnote 2: It is a property of binomial distribution that the consecutive mass isotopomer
and m3/m2 = (N-2)/3×p/q; where q = (1-p).(Reference 11)
Footnote 3: The observed consecutive mass isotopomer M1/M0 was 0.767 and
N=0.767/0.011=69.
Footnote 4 [carbon isotopomers]
[carbon isotopomers] returns a coefficient for m0=1
and for other isotopomers, coefficients = 0.
Footnote 5 N(d) is the theoretical number of deuterium atoms incorporated when
deuterated water enrichment is 100%.
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ratio is a function of N and p. It can be shown that M1/M0=Np/q , M2/M1 = (N-1)/2×p/q
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ACKNOWLEDGMENTS
The rat samples analyzed in this study were obtained from Sherin U. Devaskar who is
supported by the National Institutes of Health (HD 41230), Department of Pediatrics at
the David Geffen School of Medicine, after receiving approval from the UCLA Animal
Research Committee. Dr. G. Xiao’s current address is: Department of Biomedical
Sciences, Creighton University Medical Center, 601 N 30th ST Suite 6730, Room 6787,
Omaha, NE 68131.
This work was supported in part by PHS M01-RR00425 of the General Clinical Research
Unit, P01 AT003960-01 UCLA Center for Excellence in Pancreatic Diseases,
Metabolomics Core and a grant from the Hirshberg Foundation for Pancreatic Cancer
Research. The Voyager DE was purchased with a grant from the Henry Guenther
Foundation.
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GRANTS
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14. Papageorgopoulos C, Caldwell K, Shackleton C, Schweingrubber H, Hellerstein
MK. Measuring protein synthesis by mass isotopomer distribution analysis (MIDA).
Anal Biochem. 267(1):1-16, 1999.
15. Papageorgopoulos C, Caldwell K, Schweingrubber H, Neese RA, Shackleton CH,
Hellerstein M. Measuring synthesis rates of muscle creatine kinase and myosin with
stable isotopes and mass spectrometry. Anal Biochem. 309(1):1-10, 2002.
16. Previs F., Fatica R, Chandramouli V, Alexander JC, Brunengraber H, and Landau
BR. Quantifying rates of protein synthesis in humans by use of 2H2O: application to
patients with end-stage renal disease. Am J Physiol Endocrinol Metab 286: E665–E672,
2004.
17. Russell, W. K.; Park, Z. Y.; Russell, D. H. Proteolysis in Mixed Organic-Aqueous
Solvent Systems: Applications for Peptide Mass Mapping Using Mass Spectrometry.
Anal. Chem. 73: 2682–2685, 2001.
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isotopomer analysis. Am J Physiol. 266(3 Pt 1):E372-83, 1994.
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18. Tessari P, Kiwanuka E, Zanetti M, Barazzoni R. Postprandial body protein synthesis
and amino acid catabolism measured with leucine and phenylalanine-tyrosine tracers. Am
J Physiol Endocrinol Metab. 284(5):E1037-42, 2003.
19. Wang B, Sun G, Anderson DR, Jia M, Previs S, Anderson VE. Isotopologue
distributions of peptide product ions by tandem mass spectrometry: quantitation of low
levels of deuterium incorporation. Anal Biochem. 367(1):40-8, 2007.
20. Washburn, M. P.; Wolters, D.; Yates, J. R. III. Large-Scale Analysis of the Yeast
Proteome by Multidimensional Protein Identification Technology. Nat. Biotechnol. 19:
21. Wolfe RR. Radioactive and Stable Isotope Tracers in Medicine. New York: WileyLiss, 377–416, 1992.
22. Wu CC, MacCoss MJ, Howell KE, Matthews DE, Yates, JR. Metabolic Labeling of
Mammalian Organisms with Stable Isotopes for Quantitative Proteomic Analysis. Anal.
Chem. 76:4951-4959, 2004
23. Xiao GG, Wang M, Li N, Loo JA, Nel AE. Use of proteomics to demonstrate a
hierarchical oxidative stress response to diesel exhaust particle chemicals in a
macrophage cell line. J Biol Chem. 278(50):50781-90, 2003.
24. Yergey J. A general approach to calculate isotopic distributions for mass
spectrometry, Int. J. Mass Spectrom. Ion Phys. 52: 337-349, 1983.
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242–247, 2001.
Page 22 of 42
Page 22
FIGURE LEGENDS
Figure 1: Isotopomer clusters (m/z 1393) of peptide from rat albumin fractions. The
peptide was isolated from the albumin fractions of serum samples. The peptide was found
to be part of AMP deaminase. Spectra of the peptide from samples obtained before and
after 8 days of deuterated water treatment are shown in top and bottom panels. The
incorporation of deuterium shifts the average mass to the right (heavier) as evident by the
change in the relative peak heights.
Figure 2: Modeling of the isotope envelop with a binomial distribution. The figure insert
tryptic digest of a protein from the rat albumin fraction before deuterated water treatment.
The intensities of the individual peaks are divided by the total intensities to give
individual molar fractions. The observed isotopomers of mass peak m/z 1393 are shown
as the black bars and the distribution predicted by a binomial distribution with (N=69 and
p=0.011) is shown by the open bars. The y-axis indicates the mass shift of the
isotopomer species corresponding to molecules with 1 (m1) and 2 (m2), etc…13C
substitution. The monoisotopic species is designated as m0.
Figure 3: Definition of concatenation operation and its inverse. Concatenation operation
can be used to model the formation of “heavy” peptide from known c-isotopomers and disotopomers as shown in Figure 4. The inverse concatenation operation is used to
generate the d-isotopomer distribution given the observed isotopomer distribution of a
heavy peptide and its corresponding c-isotopomer distribution before labeling.
Figure 4: Modeling of mass shift due to deuterium incorporation. Panel A shows the
isotopomer distribution of the unlabeled peptide. M0 represents in monoisotopic
molecular species; and m1, m2 etc represents isotopomers with 1, 2, 3,... 13C
substitutions. Panel B shows the “theoretical” deuterium isotopomers which describes
the fraction of molecules containing 0, 1, 2, … deuterium atoms after labeling. The
concatenation operation returns the distribution in the observed mass isotopomers in the
labeled peptide shown in panel C. The final mass isotopomers are generated by the
presence of either 13C or 2H atoms. Panel D shows the mass isotopomers of unlabeled
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shows the “isotope envelop” of a peptide with monoisotopic mass peak at m/z 1393 from
Page 23 of 42
Page 23
(open bars) and labeled peptides (hatched bars) of the same peptide synthesized under
two different deuterium enrichments. As p(d) increases from 0.02 to 0.04, the molar
fraction of the “unlabeled” species decreases and the average molecular weight of the
peptide is shifted to the right. The differences in distribution of mass isotopomers
synthesized in the presence of deuterium enriched water is exploited to quantify the
fraction of newly synthesized protein.
Figure 5: Illustration of the inverse concatenation function and its use in determining new
protein fraction. Mass isotopomers of the “unlabeled” and “labeled” (with deuterium)
peptides are shown in the top panel. The open bars in the upper panel represent
“labeled” peptide spectrum is generated from 70% of isotopomers of panel C plus 30% of
isotopomers of panel A in Figure 4 representing 70% new synthesis. After removing the
“unlabeled” spectrum from the “labeled” spectrum using the inverse concatenation
operation, mass isotopomers resulted from deuterium incorporation is generated. The
open bars in the lower panel represent the deuterium distribution in 100% new synthesis
after determining N(d) by the m2/m1 relationship. The ratio of the mass isotopomers
(m1, m2, etc excluding m0) between the “observed” and the theoretical gives the percent
new fraction. The molar fraction of isotopomers (hatched bars) in the lower panel is 70%
of those of the open bars.
Figure 6: Linearity of mass isotopomer analysis for the determination of protein
synthesis or quantitation. The calculated percent syntheses are plotted against the
expected labeled ratios from Table 4. The linearity of the mass isotopomer analysis using
multiple linear regression analysis is demonstrated in the figure.
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isotopomers of “unlabeled” peptide and the hatched bars “labeled” peptide. The
Page 24 of 42
Page 24
TABLE LEGENDS
Table 1. Peptides identified with MALDI TOF PSD.
Tryptic digest of rat albumin was identified using post-source decay (PSD) matrixassisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (6, 7).
Table 2. Mass isotopomer distributions of the identified peptides before deuterium
labeling.
analyses. We had only one analysis for the peak at m/z 1681. Average mass was
calculated according to Blom (2).
Table 3. Mass isotopomer distributions of the identified peptides after deuterium labeling.
The averages and standard deviations were calculated from triplicate MADI-TOF
analyses. We had only one analysis for the peak at m/z 1681. Average mass was
calculated according to Blom (2). The change in average mass is given by N(d) × p ×
(New Fraction).
Table 4. Determination of percent synthesis using “labeled” and “unlabeled” spectra and
multiple linear regression analysis.
Protein fractions containing albumin and AMP deaminase from sera obtained before
deuterium labeling and on day 8 after deuterium labeling were mixed in to simulate 10,
25, 50 and 75 % synthesis. The averages and standard deviations of peptide m/z 1393
were calculated from triplicate MADI-TOF analyses for the different mixtures.
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 16, 2017
The averages and standard deviations were calculated from triplicate MADI-TOF
Page 25 of 42
Page 25
Table 5: Results from multiple linear regression analysis using an Excel
spreadsheet
program.
Using the ratio of consecutive isotopomers relationship that m2/m1 = (N(d)-1)/2 × p/(1p), we determined the theoretical N(d) to be 20. The theoretical (predicted) deuterium
isotopomers of (20, 0.025) are shown in the last column corresponding to fractions of
molecules with 0, 1, 2, 3, and 4 deuterium substitution.
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Page 26 of 42
Page 26
APPENDIX
The concatenation operation ( ) of mass isotopomers
In a molecule that contains more than one element containing isotope species, the
molecular spectrum can be considered as a complex mixture of its components. For
example the distribution of molecules affected by 13C natural abundance is approximated
by a binomial distribution with N(c) being the number of carbon and p(c) being the
reflected in the “isotope envelop”. In a protein that is synthesized in the presence of
deuterium (e.g., 4.0% deuterated water), when the contribution of other isotopes is
ignored, the distribution of molecules having 0, 1, 2, 3… deuterium substitutions is given
by the binomial distribution with N(d) being the number of possible deuterium
substitution and p(d) the deuterium enrichment in water. The isotomoper distribution in
these components are designated as (C-isotopomers) and (d-isotopomers). The protein
spectrum can be considered as the weighted sum of a series of spectra having 0, 1, 2, 3,
… deuterium substitutions, which can be derived from (C-isotopomers) and (disotopomers) using the concatenation operation ( ).
(C-isotopomers)
(d-isotopomers) = observed mass isotopomers in protein.
If distribution of isotopomer in (C-isotopomers) is represented by (a0m0, a1m1, a2m2,…)
and distribution of isotopomer in (C-isotopomers) by (b0m0, b1m1, b2m2,…). The
distribution in the protein or peptide is given by (a0b0m0, (a0b1+ a1b0)m1, (a0b2+ a1b1+
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 16, 2017
natural enrichment (1.1%). This distribution is reflected in the isotopomer distribution
Page 27 of 42
Page 27
a2b0)m2, …). M0 represents the monoisotopic species and m1, m2, m3 etc. are the
isotopomers containing 1, 2, 3, …13C or 2H isotopes. The use of mi notation allows the
comparison of peptide peaks of different masses.
(C-isotopomers) distribution is given by (a0, a1, a2, a3, …)
(d-isotopomer) distribution is given by (b0, b1, b2, b3, …)
isotopomers of the protein is given by (c0, c1, c2, c3,…)
(b0, b1, b2, b3, …) = (c0, c1, c2, c3,…)
The concatenation operation is defined by the matrix multiplication below: The cisotopomers are arranged in a (n+1 by m+1) matrix and the d-isotopomers by a (m+1 by
1) column vector. The product is the c-isotopomers in the form of a (n+1 by 1) column
vector.
a0 0 0 0 0
…
0
b0
c0
a1 a0 0 0 0 …
0
b1
c1
a2 a1 a0 0 0 …
0
b2
a3 a2 a1 a0 0 …
0
b3
c3
a4 a3 a2 a1 a0 …
0
b4
c4
an an-1 an-2
=
c2
.
.
.
.
.
.
.
.
.
…
a0
bm
cn
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 16, 2017
(a0, a1, a2, a3, …)
Page 28 of 42
Page 28
If the number of c-isotopomer is given by x, the number of c-isotopomers (x) is less than
or equal to the number of observed isotopomers in the spectrum (n > x and n > m). The
value of ai is 0 for n>i.
For a mass isotopomer distribution ci with i = n, the first matrix has the dimension of
(n+1 by m+1) of the c-isotopomers and the second matrix is a column matrix (m+1 by 1)
of the d-isotopomers. Many of the elements of these matrices may be zero as discussed
Thus,
c0 = a0b0
c1 = a0b1+ a1b0
c2 = a0b2+ a1b1+ a2b0
c3 = a0b3+ a1b2+ a2b1+ a3b0
etc.
The inverse operation
of the concatenation operation is the inverse of the above
algorithm which is to determine the d-isotopomer distribution given column matrix of
observed-isotopomers and square matrix of the c-isotopomers.
(c0, c1, c2, c3,…)
(a0, a1, a2, a3, …) = (b0, b1, b2, b3, …)
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above. The resultant (c-isotopomers) distribution is the product of these two matrices.
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Page 29
It is clear by inspection that d-isotopomer distribution can be determined using multiple
linear regression analysis. The coefficients obtained from linear multiple regression
analysis is the deuterium isotopomer distribution (b0, b1, b2, b3, …).
Using a binomial distribution with 100 carbons and 13C enrichment of 0.011, we can
show the (C-isotopmer distribution) generated for a peptide is (0.3308, 0.3680, 0.2026,
0.0736, 0.0199, 0.0042). This is the isotope envelop below deuterium incorporation.
deuterium enrichment of 0.04, the (d-isotopmer distribution) generated for a peptide is
(0.4420, 0.3683, 0.1458, 0.0364, 0.0065, 0.0009). The isotopomers in the “isotope
envelop” of the full peptide after deuterium incorporation is (0.1462, 0.2845, 0.2733,
0.1729, 0.0810, 0.0300). The effects of deuterium enrichment on isotopomer distribution
are shown in Figure 4 using the concatenation function. The proper deuterium
enrichment to be used in any experiment can be optimized using the concatenation
operation. It can be shown that the inverse operation on the isotopomers in the “isotope
envelop” of the full peptide and the pre-labeling peptide (C-isotopomers) gives the (disotopmer distribution), which shows the effect of deuterium incorporation alone.
Because of the low enrichment of deuterium used in animal studies, there is a good
likelihood that the newly synthesized peptide does not have any deuterium incorporation.
In such experiments, m0 represents pre-existing and newly synthesized peptide.
However, the fractions represented by m1 and m2 are the results of deuterium
incorporation. Thus, using the consecutive mass isotopomer equation we can determine
the N(d) and p(d), using the equation m2/m1 = (N-1)/2×p/q where q=1-p. After
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Similarly, using a binomial distribution with 20 possible deuterium positions and
Page 30 of 42
Page 30
determining deuterium enrichment, we can estimate N(d) and fraction of newly
synthesized protein by dividing the observed m1 by the theoretical m1. This is illustrated
in Figure 5.
Steps in the calculation of new fraction
We first estimated the deuterium enrichment in water to be 2.5% using deuterium
incorporation into palmitate (data not shown)(Reference 11). Using results of m/z 1393 in
of the protein using the inverse concatenation operation. The regression matrices of the
operation as described in Supplement 1 are shown below.
0.4466
0.3393
0.1448
0.0474
0.0127
0.0051
0.0000
0.4466
0.3393
0.1448
0.0474
0.0127
0.0000
0.0000
0.4466
0.3393
0.1448
0.0474
0.0000
0.0000
0.0000
0.4466
0.3393
0.1448
0.0000
0.0000
0.0000
0.0000
0.4466
0.3393
b0
b1
b2
b3
b4
=
0.2748
0.3392
0.2205
0.1025
0.0395
0.0160
Using a spreadsheet program (Excel ), the output of such a multiple linear regression is
shown below in Table 5 and the coefficients represent the distribution of mass
isotopomers due to deuterium incorporation.
Thus 61.5% of the molecules did not have any deuterium, 29.2% were labeled with one
deuterium and 7.3% labeled with 2 deuterium atoms. Using the ratio of consecutive
isotopomers relationship that m2/m1 = (N(d)-1)/2 × p/(1-p), we determined the
theoretical N(d) to be 20. The theoretical deuterium isotopomers of (20, 0.025) are
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 16, 2017
Tables 1 and 2, we set up the algorithm for the determination of deuterium isotopomers
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Page 31
(0.6027, 0.3091, 0.0753, 0.0116, 0.0013) corresponding to fractions of molecules with 0,
1, 2, 3, and 4 deuterium substitution. This is the theoretical distribution of deutertium
isotopomers in the newly synthesized protein. It can be shown by multiple linear
regression that observed distribution (X1, X2, X3 ..) accounts for 96.2% of the deuterium
distribution in m/z 1393 or the new fraction is 0.9619.
M2/m1 ratios were determined similarly for the other peptides. The corresponding
contribution of the theoretical distribution as a fraction of the observed d-isotopomer
distribution.
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theoretical distributions were calculated. The new fraction of the peptide is the
Page 32 of 42
Table 1. Peptides identified with MALDI TOF PSD
m/z
Sequences
Protein Identity
1299.4
HPDYSVSLLLR
Rat serum albumin
1393.4
MPLFKLTEIDDAM
Rat AMP deaminase
1479.5
LGEYGFQNAILVR
Rat serum albumin
1609.4
DVFLGTFLYEYSR
Rat serum albumin
1681.7
IEENGSMR
Rat alpha2u-globulin
Tryptic digest of rat albumin was identified using post-source decay (PSD) matrixassisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (6, 7).
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Page 33 of 42
Table 2. Mass isotopomer distributions of the identified peptides before deuterium
labeling
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 16, 2017
m/z 1299
m/z 1393
m/z 1479
m/z 1609
m/z 1681
M0
0.4892 ± 0.0038
0.4466 ± 0.0101
0.4239 ± 0.0029
0.3802 ± 0.0016
0.3307
M1
0.3418 ± 0.0074
0.3393 ± 0.0047
0.3349 ± 0.0056
0.3343 ± 0.0022
0.3120
M2
0.1250 ± 0.0089
0.1448 ± 0.0024
0.1480 ± 0.0030
0.1807 ± 0.0034
0.1880
M3
0.0368 ± 0.0010
0.0474 ± 0.0110
0.0581 ± 0.0056
0.0724 ± 0.0010
0.0913
M4
0.0060 ± 0.0039
0.0127 ± 0.0044
0.0247 ± 0.0025
0.0217 ± 0.0017
0.0430
M5
0.0007 ± 0.0004
0.0051 ± 0.0025
0.0105 ± 0.0038
0.0106 ± 0.0023
0.0156
1300.1348
1394.2474
1479.4711
1610.4081
1682.8889
Average
Mass
The averages and standard deviations were calculated from triplicate MADI-TOF
analyses. We had only one analysis for the peak at m/z 1681. Average mass was
calculated according to Blom (2).
Page 34 of 42
Table 3. Mass isotopomer distributions of the identified peptides after deuterium labeling
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m/z 1299
m/z 1393
m/z 1479
m/z 1609
m/z 1681
M0
0.3409 ± 0.0089
0.2748 ± 0.0044
0.2672 ± 0.0073
0.2757 ± 0.0026
0.2216
M1
0.3418 ± 0.0061
0.3392 ± 0.0090
0.3159 ± 0.0127
0.3266 ± 0.0120
0.2902
M2
0.1931 ± 0.0084
0.2205 ± 0.0063
0.2089 ± 0.0023
0.2266 ± 0.0028
0.2343
M3
0.0726 ± 0.0110
0.1025 ± 0.0028
0.1201 ± 0.0073
0.1082 ± 0.0057
0.1325
M4
0.0192 ± 0.0023
0.0395 ± 0.0014
0.0550 ± 0.0030
0.0451 ± 0.0044
0.0652
M5
0.0133 ± 0.0056
0.0160 ± 0.0022
0.0329 ± 0.0072
0.0178 ± 0.0042
0.0284
Average
1300.4942
1394.7257
1480.9391
1610.7292
1683.2361
Mass
0.3594
0.4783
0.522
0.3211
0.3472
mass
21
20
30
17
22
N(d)
New
0.6960 ± 0.0827
0.9618 ± 0.0158
0.6994 ± 0.0442
0.7380 ± 0.0131
0.6326 ± 0.0602
Fraction
The averages and standard deviations were calculated from triplicate MADI-TOF
analyses. We had only one analysis for the peak at m/z 1681. Average mass was
calculated according to Blom (2). The change in average mass is given by N(d)×p× (New
Fraction).
Page 35 of 42
Table 4. Determination of percent synthesis using “labeled” and “unlabeled”
spectra and multiple linear Regression Analysis
Unlabeled
M1
M2
M3
M4
M5
%
Synthesis
--
0.4333 ±
0.0072
0.3402 ±
0.0036
0.1574 ±
0.0014
0.0549 ±
0.0021
0.0095 ±
0.0038
0.0047 ±
0.0029
15.4 ± 6.8
25%
Labeled
0.4061 ±
0.0106
0.3360 ±
0.0222
0.1667 ±
0.0082
0.0576 ±
0.0037
0.0179 ±
0.0026
0.0157 ±
0.0094
25.6 ± 7.1
50%
Labeled
0.3550 ±
0.0106
0.3280 ±
0.0106
0.1813 ±
0.0106
0.0774 ±
0.0106
0.0309 ±
0.0106
0.0208 ±
0.0106
47.8 ± 3.5
75%
Labeled
0.3054 ±
0.0106
0.3247 ±
0.0106
0.1963 ±
0.0106
0.0958 ±
0.0106
0.0459 ±
0.0106
0.0249 ±
0.0106
68.6 ± 1.5
100% Labeled
0.2748 ± 0.0044
0.3392 ± 0.0090
0.2205 ± 0.0063
0.1025 ± 0.0028
0.0395 ± 0.0014
0.0160 ± 0.0022
--
Protein fractions containing albumin and AMP deaminase from sera obtained before
deuterium labeling and on day 8 after deuterium labeling were mixed in to simulate 10,
25, 50 and 75 % synthesis. The averages and standard deviations of peptide m/z 1393
were calculated from triplicate MADI-TOF analyses for the different mixtures.
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0.4466 ±
0.0101
0.3393 ±
0.0047
0.1448 ±
0.0024
0.0474 ±
0.0110
0.0127 ±
0.0044
0.0051 ±
0.0025
M0
10% Labeled
Page 36 of 42
Table 5: Results from multiple linear regression analysis using an Excel
program
Intercept
X Variable 1
X Variable 2
X Variable 3
X Variable 4
X Variable 5
Coefficients
0.6154
0.2919
0.0727
0.0138
0.0078
Standard Error
0.0030
0.0038
0.0038
0.0038
0.0031
t Stat
205.5862
77.6582
18.9608
3.6419
2.5213
P-value
0.0031
0.0082
0.0335
0.1706
0.2404
spreadsheet
Predicted coefficients
0.6027
0.3091
0.0753
0.0116
0.0013
Using the ratio of consecutive isotopomers relationship that m2/m1 = (N(d)-1)/2 × p/(1p), we determined the theoretical N(d) to be 20. The theoretical deuterium isotopomers
of (20, 0.025) are shown in the last column corresponding to fractions of molecules with
0, 1, 2, 3, and 4 deuterium substitution.
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Page 37 of 42
Voyager Spec #1=>BC=>NF0.7=>TR[BP = 1392.7, 24811]
1392.6913
100
2.5E+4
90
1393.6891
80
70
50
40
1394.6912
30
20
1395.6958
10
1375.6448
1379.5390
1382.6309
0
1373.0
1396.6916
1390.7392
1385.6712
1381.4
1389.8
1399.6229
1402.6671
1398.2
1405.6408
1408.6765
1412.6428
0
1415.0
1406.6
Mass (m/z)
Voyager Spec #1=>BC=>NF0.7=>TR[BP = 1393.7, 9901]
1393.6927
100
9.9E+3
90
80
1392.6939
70
1394.6969
% Intensity
60
50
40
1395.6921
30
20
1396.6938
10
1377.6168
1374.6525
0
1373.0
1405.6531
1382.6447
1380.5789
1381.4
1384.6480
1397.6768
1390.6879
1402.6187
1407.6389
1399.6308
1387.6641
1389.8
1398.2
1406.6
1410.6982
0
1415.0
Mass (m/z)
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% Intensity
60
Fig. 1
Page 38 of 42
Figure 2
Voyager Spec #1=>BC=>NF0.7=>TR[BP = 1392.7, 24811]
1392.6913
100
0.5
2.5E+4
90
1393.6891
80
0.4
% Intensity
60
50
40
1394.6912
0.3
30
20
1395.6958
10
1375.6448
0.2
0
1373.0
1379.5390
1382.6309
1381.4
1396.6916
1390.7392
1385.6712
1389.8
1399.6229
1402.6671
1398.2
1405.6408
1408.6765
1412.6428
0
1415.0
1406.6
Mass (m/z)
0.1
0
m0
m1
m2
m3
m4
m5
Mass Shift from Base Peak (m/z 1393)
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Molar Fraction
70
m6
Page 39 of 42
Figure 3
Concatenation Operation ⊕
C-Isotopomers ⊕
d-Isotopomers
Isotopomers
of peptide
Inverse Concatenation Operation ∅
∅
C-Isotopomers
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Isotopomers
of peptide
d-Isotopomers
Page 40 of 42
Figure 4
A
0.5
C-Isotopomers
N(c)=100;
p(c)=0.011
0.35
0.3
0.25
0.2
0.15
Molar Fraction
Molar Fraction
0.4
B
0.1
0.05
0.4
0.3
0.2
0.1
0
0
m0
m1
m2
m3
m4
m5
m6
m7
m0
m8
C
m1
m2
m3
m4
m5
m6
D
0.4
0.25
Molar Fraction
Observed
isotopomers
0.3
0.2
0.15
0.1
0.05
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
m0
m1
m2
m3
m4
m5
m6
m7
m8
m0
m1
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Molar Fraction
d-Isotopomers
N(d)=20;
p(d)=0.04
m2
m3
m4
m5
m6
m7
Page 41 of 42
Figure 5
0.4
Molar Fraction
0.35
Mass isotopomers of
newly synthesized
peptide from both 13C
and 2H substitutions
0.3
0.25
0.2
0.15
0.1
0.05
0
m0
m1
m2
m3
m4
m5
m6
m7
0.7
Mass isotopomers fraction
due to 0, 1, 2,… deuterium
substitutions only
70%
0.5
0.4
0.3
0.2
0.1
0
m0
m1
m2
m3
m4
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Molar Fraction
0.6
Page 42 of 42
80
y = 0.941x
2
R = 0.9744
60
40
20
0
0
20
40
60
Expected Percent Labeled
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Observed Percent Synthesis
Figure 6
80