In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic “cellular” proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present. In biomarker discovery, the detection of proteins with low abundance in the serum proteome can be achieved by optimization of protein separation methods as well as selective depletion of the higher abundance proteins such as immunoglobins (e.g. IgG) and albumin. A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatofocusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. In this study, assessment and optimization of serum separation (undepleted serum and albumin-IgG-depleted serum) by the PF2D is presented. Protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols. Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and albumin, a minimum of two peptide matches with acceptable Mascot score) in which 81 have not been identified previously in serum. Among them, numerous cellular proteins were identified to be specifically the skeletal muscle isoform, such as skeletal muscle fast twitch isoforms of troponin T, myosin alkali light chain 1, and sarcoplasmic/endoplasmic reticulum calcium ATPase. The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic “cellular” proteins as biomarkers without further characterization of the precise isoforms or post-translational modifications present. There has been a surge of interest in the proteomic analysis of plasma and serum in the search for clinically relevant biomarkers of disease. In biomarker discovery, it is necessary to maximize the observation of the plasma or serum proteome to detect proteins with low abundance. This can be achieved by optimization of protein separation methods as well as selective depletion of the proteins at high abundance such as immunoglobins (e.g. IgG) and albumin. There are a large number of proteomic technologies that separate either proteins or peptides prior to MS (1Graham D.R. Elliott S.T. Van Eyk J.E. Broad-based proteomic strategies: a practical guide to proteomics and functional screening.J. Physiol. 2005; 563: 1-9Google Scholar). A relative newcomer to the proteomic separation arena is the commercial instrument PF2D from Beckman Coulter that separates proteins in the first dimension using chromatographic focusing followed in line by reversed phase chromatography in the second dimension, thereby separating intact proteins based on pI and hydrophobicity. To date, assessment and optimization of either plasma or serum separation by the PF2D has not been undertaken. Generally LC proteomic methods have focused on separating complex mixtures of peptides obtained following digestion of the serum proteome (peptide LC, shotgun), whereas separation of proteins has been relegated primarily to electrophoresis in both one and two dimensions (2DE) 1The abbreviations used are: 2DE, two-dimensional electrophoresis; PTM, post-translational modification; TnT, troponin T; sTnT, skeletal troponin T; TnI, troponin I; sTnI, skeletal troponin I; CF, chromatofocusing. (2Swanson S.K. Washburn M.P. The continuing evolution of shotgun proteomics.Drug Discov. Today. 2005; 10: 719-725Google Scholar, 3Tsikas D. Caidahl K. Recent methodological advances in the mass spectrometric analysis of free and protein-associated 3-nitrotyrosine in human plasma.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2005; 814: 1-9Google Scholar). 2DE has an advantage over peptide-based LC methods as it enhances the ability to identify the precise isoforms of proteins that are present and/or post-translational modifications (PTMs) that may alter the pI or mass of a protein (1Graham D.R. Elliott S.T. Van Eyk J.E. Broad-based proteomic strategies: a practical guide to proteomics and functional screening.J. Physiol. 2005; 563: 1-9Google Scholar). The potential to separate proteins, rather than peptides, by liquid chromatography using two dimensions would potentially provide the same advantages as 2DE. The PF2D system (Beckman Coulter) is a two-dimensional LC system that uses chromatographic focusing to separate intact proteins in the first dimension by pI (from 8.5–4.0) and in the second dimension by reversed phase chromatography, which separates based on hydrophobicity, thus enhancing the precise detection of isoforms and/or PTMs that alter the pI and/or hydrophobicity of a protein. Also by resolving proteins based upon their intrinsic characteristics prior to mass spectrometry analysis into fractions containing a few proteins (one up to 20), a high degree of sequence coverage for each identification can be obtained to observe PTMs. This is in contrast to shotgun databases in which protein identifications can be made on a single observed peptide with no information about the nature of the parent protein. In this study, we outline the optimization of the solubilization and separation conditions for the human serum proteome on the PF2D. To assess the utility of the PF2D, protein databases were created for serum obtained from a healthy individual under traditional and optimized methods and under different sample preparation protocols to remove lipids, albumin, and immunoglobulins. 100 μl of serum was delipidated and depleted sequentially of albumin and IgG (doubly depleted serum) as outlined in Fu et al. (4Fu Q. Garnham C.P. Elliott S.T. Bovenkamp D.E. Van Eyk J.E. A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis.Proteomics. 2005; 5: 2656-2664Google Scholar). Briefly serum was centrifuged for 15 min at 15,000 × g, and the lipid-containing upper layer was removed. The delipidated serum was mixed with protein G-Sepharose beads (HiTrap protein G HP, Amersham Biosciences) in 100 mm NaCl, 10 mm HEPES, pH 7.4 and mixed in a Handee Mini-spin column (Pierce) for 1 h at room temperature and then centrifuged (6000 rpm for 3 min) to pellet beads. Prechilled 95% ethanol (Sigma) was added to the supernatant to a final concentration of 42% and incubated for 1 h at 4 °C with gentle mixing followed by centrifugation at 16,000 × g for 45 min at 4 °C. The supernatant (albumin-enriched fraction) was removed, and the pellet (serum proteins depleted of albumin) was stored at −80 °C. Over 95% of the albumin and IgG is removed (4Fu Q. Garnham C.P. Elliott S.T. Bovenkamp D.E. Van Eyk J.E. A robust, streamlined, and reproducible method for proteomic analysis of serum by delipidation, albumin and IgG depletion, and two-dimensional gel electrophoresis.Proteomics. 2005; 5: 2656-2664Google Scholar). Purified bovine serum albumin (Sigma), the pellet (doubly depleted serum), or unfractionated serum was suspended in PF2D start buffer (pH 8.5). Protein concentrations were determined in duplicate by the BCA protein assay (Pierce). The albumin, serum, or the doubly depleted serum was analyzed on a one-dimensional liquid chromatography system (System Gold system with autoinjector, Beckman Coulter) or a two-dimensional liquid chromatography system (ProteomeLab PF2D, Beckman Coulter). The serum analysis was exclusively carried out on new naïve columns (which were not used for any other experiments) with strong wash conditions between runs to ensure no contamination from another analysis. The first dimension of PF2D consists of a single piston pump (HPCF Module), manual injector for sample introduction, pH monitor, and UV detector. The first dimension separation consists of chromatofocusing based on charge. Fractions from the first dimension are collected in the fraction collector/injector (FC/I Module), which is the interface between the first and second dimensions. Fractions are automatically introduced into the second dimension reversed phase chromatography, which separates based on hydrophobicity. The second dimension consists of a binary pump system (HPRP Module), column heater, UV detector, and fraction collector. The fractions are collected into 96-deepwell plates for mass spectrometry analysis. The hardware is controlled by 32 Karat software. With this system, the first and second dimensions occur sequentially in an automatic manner. Chromatofocusing was carried out on the CF column by mixing two buffers with different pH, Start Buffer (pH 8.5) and Eluent Buffer (pH 4.0), to create a linear pH gradient from 8.5 to 4.0 that is followed by a wash buffer comprising 1 m NaCl. 1 mg of purified bovine albumin, 1.5 mg of depleted serum, or 3 mg of native undepleted serum was injected onto the CF column equilibrated for 130 min at 0.2 ml/min with the proprietary start buffer that included urea and a reducing agent at pH 8.5. The pH gradient was achieved by introducing increasing amounts of the eluent buffer (pH 4.0, either 0, 10, or 20% acetonitrile, methanol, or isopropanol) at a flow rate of 0.2 ml/min over 95 min. The CF column was then washed for 45 min with the third buffer containing 1 m NaCl and re-stored in water. Fractions were collected every 5 min except during the pH gradient portion of the run when fractions were collected at 0.3 pH intervals. Each fraction (200–500 μl) was sequentially analyzed by reversed phase HPLC at a constant 50 °C. Proteins were separated on a non-porous C18 reversed phase column using 3.33% B/min linear gradient in which solvent A was 0.1% aqueous TFA and solvent B was 0.08% TFA in acetonitrile at a flow rate of 0.75 ml/min. Proteins were monitored at 214 nm. The reversed phase fractions were collected by 0.25 min/tube and stored at −80 °C for further analysis. For one-dimensional LC, samples were separated on the same column as used in the second dimension of PF2D with the same gradient and flow rate. The percentage of albumin eluted on different columns and at different pH conditions was determined by calculating the peak area of the albumin monitored at 214 nm, a wavelength at which the peak area is directly proportional to the quantity of the protein(s) (5Van Eyk J.E. Thomas L.T. Tripet B. Wiesner R.J. Pearlstone J.R. Farah C.S. Reinach F.C. Hodges R.S. Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament.J. Biol. Chem. 1997; 272: 10529-10537Google Scholar, 6Tripet B. Van Eyk J.E. Hodges R.S. Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their in the Ca2+-dependent of muscle Biol. 1997; Scholar). spectrometry was carried out to the of the albumin The reversed phase fractions μl) obtained from the two-dimensional LC were using a to 1 m was added to the to samples to pH at an of was added and incubated at °C was added to the of peptides was on an instrument at The and Proteins identified with obtained from a minimum of two peptide matches with a minimum Mascot of for each peptide protein identifications were made with two peptide matches the to be to the protein not to other potential identifications in the of serum analysis using a protein was to be present in the was identification based on a single and are in was added by any protein that be to than one protein. To create a non-redundant the protein identifications were in the for and numerous were the same protein was in protein of a protein or proteins were based on a peptide to a sequence of the or intact protein. The pI and mass were from the for protein identification were to ensure Proteins be between their and were no peptide observed by MS that to an sequence to the The of a protein is a peptide was observed by MS that to an sequence to the specific for whereas optimized serum isoform, skeletal identified in two different 3 isoform, skeletal identified in two different identified based on single peptide identified in two different identified in two different identified in two different chain identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different 1, 50 chain to specific of identified in two different identified in two different identified in two different identified based on single peptide identified in two different 1 identified based on single peptide identified in two different identified based on single peptide 1 1, identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different B identified in two different identified in two different identified in two different I light I identified in two different identified in two different muscle 1 identified in two different identified in two different protein identified in two different identified in two different identified in two different 1 identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different chain chain identified in two different protein identified in two different identified in two different identified based on single peptide identified in two different identified in two different of 1, skeletal muscle, alkali light chain 1 skeletal fast alkali light chain 1, skeletal twitch identified in two different protein identified in two different protein identified based on single peptide identified in two different identified based on single peptide identified in two different identified in two different identified in two different identified in two different identified in two different protein protein protein reticulum calcium ATPase skeletal fast twitch muscle 1 identified in two different identified in two different identified in two different A protein to identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different identified in two different skeletal fast T, fast skeletal calcium identified in two different identified in two different identified in two different identified based on single peptide identified in two different identified in two different Protein identification were to ensure Proteins be between their and were no peptide observed by MS that to an sequence to the The of a protein is a peptide was observed by MS that to an sequence to the specific for whereas Protein identified in two different Protein identified based on single peptide in a new With the PF2D running conditions by the for plasma Beckman we were to identify a of non-redundant proteins, any in serum from a healthy human individual proteins have not been previously in the non-redundant by et al. R.S. The human plasma a by of separate Scholar). are based on the detection of proteins by two or the search or a proteomic analysis using two-dimensional gel electrophoresis of plasma Q. S.T. The human serum of separated protein on two-dimensional electrophoresis and identification of or following of the plasma proteome analysis by R.J. a human serum analysis by separation with mass Scholar, R.S. of the low human serum to proteins by a single of identification with either human serum or plasma and included from et al. R.J. proteome of human plasma following in using with mass 2005; 5: which were the et al. R.S. The human plasma a by of separate In proteins have not been by any proteomic method to both traditional serum proteins as well as proteins of cellular In the identification of a was to it a For the 3 was unambiguously identified in the doubly depleted serum using the PF2D whereas the in human plasma been previously R.S. The human plasma a by of separate Scholar, R.J. proteome of human plasma following in using with mass 2005; 5: Scholar). serum proteins were eluted in two at their pI and with the or were eluted during the wash the wash is to proteins with a pI 4.0, the pI of the proteins in the wash from to (e.g. and proteins not in this fraction running conditions under running conditions by the albumin a and eluted in the first dimension fraction collected between pH and and in the wash This that either is of protein during the solubilization and/or binding of proteins to the first dimension To both we both solubilization and first dimension running conditions conditions are in and the of albumin either from human serum or purified albumin which in a and was eluted in two fractions using the method not of and/or solvent to the solubilization buffer was with optimized first dimension separation included the of 20% isopropanol to first dimension running which between proteins and the CF The of 20% isopropanol the percentage of albumin from the CF column in the pH was a in the This is by the area of the albumin peak 1, in the pH and fractions when the serum sample was separated using the method or when 20% isopropanol was present in the first dimension The peak at min albumin based on the analysis of the reversed phase fraction and is with an in The is in the serum sample run under the method in which was a of present in the albumin peak in the optimized conditions with 20% isopropanol present in the first dimension was not present. The of 20% isopropanol on the of serum proteins was to the number of proteins unambiguously identified to it the number of proteins in two fractions the proteins in two fractions I, with that are at their pI I, this the quantity of proteins in the wash was based on the of the sequence coverage R.J. proteome of human plasma following in using with mass 2005; 5: of chromatographic focusing conditions for separation of albumin present in human for solubilization and eluent in eluted at eluted in percentage of with buffer with buffer acetonitrile in eluent acetonitrile in eluent in eluent isopropanol in eluent isopropanol and buffer with buffer isopropanol in eluent isopropanol in eluent high pH in a new Separation of the doubly depleted serum using the PF2D with 20% isopropanol present in the first dimension running buffer allowed us to unambiguously identify 150 non-redundant serum proteins (excluding all immunoglobulin and an of proteins (which primarily low abundance proteins and cellular with separation under the same conditions of undepleted serum from the same In of the 150 non-redundant serum proteins, 81 have not been identified previously by two or outlined by et al. R.S. The human plasma a by of separate in the serum is of serum proteins, cellular proteins, and proteins with no or that are from or proteins 1 for of the identification of of of of all the proteins observed by the PF2D analysis been to be in serum, not from a single R.S. 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The the of non-redundant is to that proteins the plasma were identified based on two or This is of their and to the number of proteins we have unambiguously identified with a protein identification or a et al. R.J. proteome of human plasma following in using with mass 2005; 5: analyzed 3 mg of peptides obtained from the digestion of μl of undepleted plasma from healthy and The peptides were separated by dimension proteins were identified based on than one peptide from any of LC potential for the higher number of proteins is the of proteins with which a strong phase the of proteins that were identified have plasma concentrations 10 one of their proteins was a serum protein with which has an plasma concentration in healthy individuals R.J. proteome of human plasma following in using with mass 2005; 5: Scholar). the PF2D to separate intact proteins with optimized and depletion of the serum has allowed the detection of serum proteins such as which has an plasma concentration of 10 The concentration of cellular proteins with low abundance is to assess as the plasma concentration in has not been it is to an of skeletal troponin which we based on of skeletal troponin I a protein that in a concentrations in plasma of healthy individuals has been et al. D. B. troponin I as a of muscle Physiol. 1997; Scholar, B. B. of muscle proteins running in and Sci. to be and then between with and in healthy and we concentrations in healthy individuals and with muscle to be of assay R.J. Van Eyk J.E. and skeletal troponin I in serum from with skeletal muscle a Chem. 2005; Scholar). The of in is an of the sensitivity that can be achieved using the PF2D under optimized There is an between a proteomic method that is to detect low abundance proteins with concentrations in the is to identify The for this low number of proteins identified the high degree of used for protein Protein identifications were made based on two or peptides that unambiguously to a single protein. was to ensure with to other protein and the of or proteins were analyzed for sequence and were matches was the for peptide as the relative abundance of a protein are not out the large serum databases are based on which the number of This the for for The of cellular of proteins in the serum of healthy individuals has in biomarker For the of both and in serum is a for the of Generally biomarkers from cellular proteins one of the following the in serum to a or the between a and a and the of a or The of skeletal muscle proteins into the proteins from any a that be to the ability to unambiguously identify specific isoforms or PTMs will the number of proteins that can be The PF2D system is to separate the serum proteome to the detection of proteins with high and low abundance. The of 20% isopropanol to the first dimension buffer enhances the of proteins at or their The detection of specific skeletal muscle protein isoforms in the serum from healthy individuals reflects the physiological turnover that occurs in skeletal muscle, which will have an impact on the ability to use generic “cellular” proteins as biomarkers without further characterization of the precise isoforms or present.
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