From the
Proteomics Section, Molecular Probes,
Inc., Eugene, Oregon 97402-9144 and the
¶Institute of Molecular Biology, University of
Oregon, Eugene, Oregon 97403-1229
Received for publication, May 2, 2003 , and in revised form, May 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recently, a detailed human heart mitochondrial proteome data base of 615 proteins was generated using a combination of our sucrose gradient fractionation and SDS-polyacrylamide gel electrophoresis method (16), followed by mass spectrometry (17). We demonstrate an important extension of this approach for the comprehensive analysis of the mitochondrial phospho-proteome, using the sucrose gradient fractionation/gel electrophoresis separation approach in concert with fluorescence-based multiplexed proteomics staining technology and mass spectrometry (16, 18, 19). Mitochondrial multisubunit protein complexes are first separated by size using sucrose gradient centrifugation, and the individual subunits are then resolved by one-dimensional SDS-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. Subsequently, gels are fluorescently stained and imaged to reveal phosphorylation levels using a fluorescent phosphosensor dye (Pro-Q Diamond dye) followed by staining and imaging to reveal protein expression levels using a fluorescent total protein indicator (SYPRO® Ruby dye (Molecular Probes, Inc.)) (1921). Finally, peptide mass fingerprinting by MALDI-TOF1 mass spectrometry is employed to identify the phosphoproteins revealed by the dichromatic staining technique. This study is the first successful application of Multiplexed Proteomics technology to the discovery of novel phosphoproteins in a complex biological specimen.
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EXPERIMENTAL PROCEDURES |
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Phosphoprotein Separation and Detection ProceduresSDS-polyacrylamide gel electrophoresis was performed by standard methods (23). Proteins were concentrated using a chloroform/methanol precipitation procedure (24) before resuspension in sample buffer and heating for 5 min at 95 °C. Samples were cooled to room temperature before gel loading and electrophoresis. For two-dimensional gel electrophoresis, mitochondrial proteins were prepared as described previously (16). All samples were precipitated before two-dimensional gel electrophoresis to minimize unspecific staining due to phospholipids and other cell constituents. Approximately 100150 µg of protein was separated for 80,000 V-h on pH 310 Immobiline Drystrip-immobilized pH gradient gels (Amersham Biosciences). After isoelectric focusing, SDS-polyacrylamide gel electrophoresis was performed using an Investigator two-dimensional system (Genomic Solutions, Ann Arbor, MI).
Fluorescent staining of SDS-polyacrylamide gels using Pro-Q Diamond phosphoprotein gel stain (Molecular Probes, Eugene, OR) was performed by fixing the gels in 45% methanol, 5% acetic acid overnight, washing with three changes of deionized water for 10 to 20 min per wash, followed by incubation in Pro-Q Diamond phosphoprotein gel stain for 180 min, and destaining with successive washes of 15% 1,2-propanediol or 4% acetonitrile in 50 mM sodium acetate, pH 4.0. Useful images could be obtained 3 h after staining, employing three successive destaining washes. Following image acquisition, gels were stained for total protein with SYPRO Ruby protein gel stain (Molecular Probes) for serial dichromatic detection, permitting comparison of phosphoprotein and total protein profiles (20, 21).
Images were acquired on a Fuji FLA 3000 laser scanner (Fuji Photo Film Co., Ltd., Tokyo, Japan) with 532 nm excitation and 580 nm band pass emission filter for Pro-Q Diamond dye detection and with 473 nm excitation and 580 nm band pass emission filter for SYPRO Ruby dye detection. For two-dimensional gels, computer-generated differential display maps of protein phosphorylation and protein expression patterns were generated using Z3 software (Compugen, Tel-Aviv, Israel) (25). With this system, spots from the reference gel appear green and those from the comparative gel appear magenta. When images are aligned, similarly intense spots in the overlay image appear gray or black, while those that differ in intensity levels appear green or magenta. This facilitates identification of differentially expressed protein spots by simple visual inspection.
Matrix-assisted Laser Desorption Time-of-Flight Mass SpectrometryAfter detecting proteins in polyacrylamide gels with Pro-Q Diamond phosphoprotein gel stain and staining with SYPRO Ruby protein gel stain, protein bands were subjected to trypsin digestion and mass spectrometry, as described previously (19, 26). Mass spectrometry was performed using an Axima CFR MALDI-TOF mass spectrometer (Kratos Analytical, Chestnut Ridge, NY) with an accelerating voltage of 20 kV, and profiles were internally calibrated using trypsin auto-proteolytic fragments. Peptide-mass fingerprinting data were evaluated using the Kratos Launchpad analysis package and the Protein Prospector data base.
Western BlottingLarge format one-dimensional gels were electro-blotted according to published protocols (27). Phosphoamino acid-specific antibodies were visualized by chemiluminescence using an ECL kit (Amersham Biosciencs). The anti-phosphoserine antibody was from Zymed Laboratories Inc. (South San Francisco, CA), whereas the anti-phosphotyrosine and anti-phosphothreonine antibodies were from Cell Signaling Technology (Beverly, MA).
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RESULTS AND DISCUSSION |
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Fig. 1 shows a
representative SDS-polyacrylamide gel of the sucrose gradient fractions
19, as well as unfractionated bovine heart mitochondria, stained with
Pro-Q Diamond dye (Fig.
1A) and then subsequently with SYPRO Ruby dye
(Fig. 1B). Several
proteins are obviously stained with the phosphoprotein-selective dye, and
these proteins do not in general correspond to the most abundant proteins in
the gradient fractions. As an example of the differential staining, the gel
lane containing fraction 2 of the sucrose gradient was investigated further
using image analysis software. Electrophoretic profiles were obtained for the
lane after Pro-Q Diamond dye staining and then after SYPRO Ruby dye staining.
The overlay of the two profiles (Fig.
2) demonstrates that a 40-kDa protein is stained
significantly by the phosphate-selective dye. There was little or no staining
of the other proteins present in this lane, confirming the earlier studies
showing that the background of nonspecific labeling of unphosphorylated
proteins is low (19). The
treatment of phosphoproteins or phosphopeptides with a strong base (0.1
M Ba(OH)2) eliminates phosphoric acid from phosphoserine
and phosphothreonine residues through a
-elimination reaction
(28).
-Elimination
experiments confirmed phosphorylation of the protein from gradient fraction 2.
Using in-gel barium hydroxide treatment at 37 °C for 1 h, led to loss of
up to 50% of the Pro-Q Diamond dye binding to the
40-kDa protein compared
with an untreated control gel, whereas no Pro-Q Diamond signal was obtained
from ovalbumin after
-elimination of the serine phosphate residues (data
not shown).
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As a first operational screen, a Pro-Q Diamond dye-to-SYPRO Ruby dye
fluorescence ratio (D/S) that was 1.5 times the averaged ratio obtained with
the nonphosphorylated molecular mass markers bovine serum albumin,
phosphorylase b, carbonic anhydrase, and -galactosidase (in
this case 0.36) was used to define phosphoproteins in gels of sucrose gradient
fractionated mitochondrial extracts. All ratios were corrected for molecular
mass, since a protein with 100 kDa mass will bind more SYPRO Ruby dye
molecules than a 50-kDa protein and result in a 50% lower D/S ratio for the
larger protein. By the cited criterion, there were five prominent
phosphoproteins revealed in the SDS-polyacrylamide gel analysis indicated in
Fig. 1.
Identification of the 42-kDa Protein as a Novel Phosphoprotein in
Complex ITo further investigate the phosphorylation of the
40-kDa protein, sucrose gradient fraction 2 was separated by
two-dimensional gel electrophoresis, the spots excised, digested with trypsin,
and subjected to mass spectrometry analysis. The peptide mass profile
identified the protein unequivocally as the bovine homologue of human NDUFA10
of Complex I, the NADH:ubiquinone oxidoreductase. Complex I catalyzes the
first step of the electron transport chain, the transfer of two electrons from
NADH to ubiquinone, coupled to the translocation of four electrons across the
membrane. It is found in the inner mitochondrial membrane as an assembly with
molecular mass of over 900 kDa and consists of
46 subunits
(29,
30), seven being encoded by
mitochondrial DNA, while the remaining are encoded by nuclear DNA.
The identification of NDUFA10 was further confirmed and extended in two-dimensional gel electrophoresis experiments. The phosphorylated protein migrated to a similar position as the 42-kDa protein (NDUFA10) annotated on a previously published map of bovine complex I (29). As shown in Fig. 3, SYPRO Ruby dye staining revealed five isoforms for the protein (spots 15), three of which were phosphorylated, based upon Pro-Q Diamond dye staining. The most straightforward interpretation of the staining patterns of the NDUFA10 isoforms is as follows: spots 1 and 2 are two unphosphorylated forms, different in charge through amino acid differences or post-translational modifications other than phosphorylation. Spots 3 and 4 are the phosphorylated forms of each or are the mono- and diphosphorylated forms of one of them. Spot 5 is a phosphorylated form of a third variant of the polypeptide, whose nonphosphorylated form is buried in spot 4. Notably, the intensity ratio of phosphate-to-protein in spot 4 is close to twice that in spot 3. As the Pro-Q Diamond dye signal intensity correlates with the number of phosphate residues on a protein (19), it appears that the isoform in spot 4 is doubly phosphorylated and in spot 3 singly phosphorylated. Based upon Western blot analysis (Fig. 4) NDUFA10 contains only phosphothreonine residues, while a cAMP phosphorylation motif scan of the polypeptide sequence using the ProSite data base program (31) identified two threonine phosphorylation motifs, KKmT and KKvT. No potential sites of protein kinase A-mediated serine phosphorylation were uncovered by this analysis.
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The 18-kDa subunit of complex I (human NDUFS 4) has previously been
reported to be phosphorylated by a cAMP-dependent protein kinase using
exogenously added kinase and [-32P]ATP or by
anti-phosphoserine immunoblotting of proteins from mitoplasts of activated
cells
(57,
32). Phosphorylation of this
subunit was not observed here using the described Multiplexed Proteomics
technology (see Fig. 2),
suggesting that the protein is not typically phosphorylated to a significant
extent in resting mitochondria.
Other proteins besides NDUFA10 for which the ratio of Pro-Q Diamond dye
staining to SYPRO Ruby dye staining was more than 1.5 times the average
background are listed in Table
I. These are adenine nucleotide translocase (ANT), isocitrate
dehydrogenase, flavoprotein of succinate dehydrogenase, NAD(P)
transhydrogenase, aconitase, and the phosphate carrier protein. The other
proteins that reacted significantly with the Pro-Q Diamond stain, but did not
produce the 1.5 ratio of phosphate to protein stain used in the first cut off,
are listed in Table I. These
include and
subunits of complex V, core proteins I and II of
complex III, and creatine kinase. A phosphoprotein of
40 kDa in sucrose
gradient fractions 7 and 8 was identified by monoclonal antibody reaction as
the E1
subunit of pyruvate dehydrogenase, but the identity of this
protein could not be confirmed by mass spectrometry, probably because the
bovine sequence is significantly different from the human sequence used in the
analysis. This latter group of proteins probably represents those for which
only a fraction of the total are phosphorylated in the steady state under
conditions present in heart tissue. It is important to note that some of the
proteins listed in Table I have
previously been identified as phosphoproteins
(9,
10). However, the major
phosphoproteins described here, NDUFA10 and ANT, have not been reported
previously.
In summary, we describe the first use of multiplexed proteomics technology on a complex mixture of proteins, bovine heart mitochondria, and establish the utility of the protocols for identifying phosphoproteins in one-dimensional and two-dimensional gels. Importantly it is clearly possible to distinguish between phosphorylation of the different isoforms of a polypeptide with this technology. Here we focus on NDUFA10 of Complex I, whose identification as a phosphoprotein is novel and intriguing. The phosphorylation of this subunit could serve two distinct functions. One might be to control the activity of Complex I through binding of NADH (33). Additionally, since it has been noted that the NDUFA10 subunit of Complex I is easily lost during purification of complex I, the phosphorylation might influence the binding affinity of NDUFA10 and in turn regulate the amount of fully active Complex I in the inner-membrane of mitochondria.
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FOOTNOTES |
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Both authors contributed equally to the work.
|| To whom correspondence should be addressed: Proteomics Section, Molecular Probes, Inc., 29851 Willow Creek Rd., Eugene, OR 97402. Tel.: 541-984-5692; Fax: 541-344-6504; E-mail: wayne.patton{at}probes.com.
1 The abbreviations used are: MALDI-TOF, matrix-assisted laser desorption
ionization time-of-flight; ANT, adenine nucleotide translocase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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