Article pubs.acs.org/jpr
Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry Dong Huey Cheon,†,‡ Eun Ji Nam,†,§ Kyu Hyung Park,∥ Se Joon Woo,∥ Hye Jin Lee,§ Hee Cheol Kim,⊥ Eun Gyeong Yang,† Cheolju Lee,†,# and Ji Eun Lee*,† †
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, Republic of Korea § Department of Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea ∥ Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam 463-707, Republic of Korea ⊥ Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea # Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea ‡
S Supporting Information *
ABSTRACT: While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis. KEYWORDS: human plasma, low-molecular-weight (LMW) plasma proteins, cleaved products, top-down mass spectrometry, gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), post-translational modification (PTM), single amino acid variation (SAAV), biomarker, colorectal cancer (CRC)
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laser desorption/ionization (SELDI)-TOF MS.7,8 In the comparative mass spectrometric profiling of blood plasma/ serum samples, many of the species that were significantly changed between control and disease samples were shown to have low molecular masses. The discovery of LMW species as potential biomarkers was made as a consequence of the bias of MALDI-TOF MS in the detection of LMW peptides and proteins; however, the profiling approach led to the discovery of the potential of LMW components present in plasma or serum as a rich source of diagnostic biomarkers, which were often overlooked by traditional techniques such as 2D gel electrophoresis.9 Because the initial discovery of potential serum or
INTRODUCTION
Human blood plasma or serum serves as a great source for disease diagnosis because it is readily sampled and has a wealth of diagnostic information regarding the state of the human body in health and disease.1,2 While plasma or serum proteome is known to contain valuable biomarkers to differentiate normal and pathological states, the low-molecular-weight (LMW) species (i.e., 2.0-fold decreases. Specifically, APOC1 and most of the truncated products exhibited ≥1.5-fold decreases in the CRC plasma sample. The APOC1 level was previously found to be higher in preoperative serum than in postoperative serum obtained from pancreatic cancer patients;67 however, many other studies showed that the protein level was reduced in cancer serum samples, including breast cancer, papillary thyroid carcinoma, and stomach cancer.68−70 In addition, Engwegen et al. reported that the APOC1 level decreased in serum samples of CRC patients compared with those of controls and adenomatous polyps patients from their SELDI-TOF analysis.71 As for the TTR, the levels of protein and its proteoform with SAAV from Cys30Gly were found to decrease 2.0-fold, respectively, in the CRC plasma sample, whereas the S-sulfonated form exhibited a 1.5-fold increase in the CRC plasma sample. (See Figure 4D of the relative abundances of the SAA1 proteoforms.) When we summed all of the peak area values for TTR and the proteoforms and calculated the relative abundance ratio based on the peak area values, the TTR proteoforms exhibited >2.0-fold decreases in the CRC plasma sample. While TTR binds and transports the thyroid hormones and the retinol-binding protein-retinal complex,72 it has been found that different levels of TTR and the proteoforms in serum is linked to several cancers, including lung, ovarian, and endometrial cancer.73−75 It was also found that the levels of TTR and its proteoforms with S-cysteinylation and S-glutathionylation decreased in serum samples from colorectal adenoma and CRC patients,76 which correlates with our results for the TTR abundance in the CRC plasma sample. While several previous reports support our quantitative results obtained from top-down mass spectrometric analysis, we performed Western blot experiments for the six LMW proteins that showed >1.5-fold changes between the HC and CRC plasma samples to further confirm the mass spectral results. The levels of PF4, CXCL7, SAA1, CCL14, APOC1, and TTR were examined via Western blotting, and APOA1, exhibiting a 1.1-fold change between the HC and CRC plasma samples, was also included for Western blot analysis. As seen in Supporting Information Figure S13 and Figure 4E,F, the four LMW proteins (PF4, CXCL7, SAA1, and CCL14) were expressed at higher levels in the CRC plasma sample, whereas the APOC1 and TTR levels were reduced in the CRC plasma sample. The APOA1 level between the two plasma samples was also found to be similar, implying
that the Western blot results correlate with the quantitative results obtained from top-down mass spectrometric analysis. Although the Western blot results examined for the six proteins showing >1.5-fold changes between the HC and CRC plasma samples correlated with the mass spectral results, further analysis using biological replicates is needed to confirm the observed changes between the two plasma samples are significant. While we did not perform comparative analysis using the HC and CRC plasma samples with immunoaffinity depletion in the current study, we think that the current top-down platform can be applied to the comparative analysis of the plasma samples with immunoaffinity depletion; however, in the case of the plasma samples with immunoaffinity depletion, the depletion process needs to be well controlled so that the immunoaffinity depletion efficiency can be similar to the samples used for quantitative analysis. Because the top-down mass spectrometry allows quantitation of cleaved products of the higher molecular weight proteins, it is expected that the extent of immunoaffinity depletion of high-abundance proteins will also affect the quantitation of the cleaved products from the higher molecular weight proteins. In the present study, we also had to run extra LC−MS/MS runs prior to LC−MS analyses for the purpose of identification and then calculated the relative abundance ratios of the peak areas using LC−MS data to acquire more accurate quantitative information; however, it is expected that a state-ofart mass spectrometer with faster duty cycle can allow both identification and quantitation using the LC−MS/MS data.
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CONCLUSIONS The current top-down mass spectrometric analysis of LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins allowed for identification and characterization of both LMW proteins and cleaved products of larger proteins, including those with PTMs and SAAVs. Specifically, from the depleted plasma samples, identification of cleaved products from the high-abundance proteins, which were supposed to be removed from the immunoaffinity depletion, largely decreased, facilitating identification of the LMW proteoforms derived from more different kinds of proteins, including those with lower abundances. When comparative analysis for plasma samples without removal of high-abundance proteins between HCs and CRC patients was further pursued using top-down mass spectrometry, tens of LMW proteoforms changed >1.5-fold between the HC and CRC plasma samples by calculating the ratios of peak areas from the XICs. The Western blot results examined for the six proteins were also consistent with the mass spectral results. Although the LMW proteoforms exhibiting level changes between the HC and CRC plasma samples in the current study need to be validated using a large number of biological replicates, we believe that the current top-down platform can be used to discover candidate biomarkers that show differential abundance levels or undergo PTMs and SAAVs in disease states.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00773. Figures S1−S13 include slab gel visualizations of the GELFrEE fractionation, Venn diagram showing the M
DOI: 10.1021/acs.jproteome.5b00773 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
■
(12) Greening, D. W.; Simpson, R. J. A centrifugal ultrafiltration strategy for isolating the low-molecular weight (
Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry Dong Huey Cheon,†,‡ Eun Ji Nam,†,§ Kyu Hyung Park,∥ Se Joon Woo,∥ Hye Jin Lee,§ Hee Cheol Kim,⊥ Eun Gyeong Yang,† Cheolju Lee,†,# and Ji Eun Lee*,† †
Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742, Republic of Korea § Department of Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea ∥ Department of Ophthalmology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam 463-707, Republic of Korea ⊥ Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea # Department of Biological Chemistry, University of Science and Technology, Daejeon 305-333, Republic of Korea ‡
S Supporting Information *
ABSTRACT: While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis. KEYWORDS: human plasma, low-molecular-weight (LMW) plasma proteins, cleaved products, top-down mass spectrometry, gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), post-translational modification (PTM), single amino acid variation (SAAV), biomarker, colorectal cancer (CRC)
■
laser desorption/ionization (SELDI)-TOF MS.7,8 In the comparative mass spectrometric profiling of blood plasma/ serum samples, many of the species that were significantly changed between control and disease samples were shown to have low molecular masses. The discovery of LMW species as potential biomarkers was made as a consequence of the bias of MALDI-TOF MS in the detection of LMW peptides and proteins; however, the profiling approach led to the discovery of the potential of LMW components present in plasma or serum as a rich source of diagnostic biomarkers, which were often overlooked by traditional techniques such as 2D gel electrophoresis.9 Because the initial discovery of potential serum or
INTRODUCTION
Human blood plasma or serum serves as a great source for disease diagnosis because it is readily sampled and has a wealth of diagnostic information regarding the state of the human body in health and disease.1,2 While plasma or serum proteome is known to contain valuable biomarkers to differentiate normal and pathological states, the low-molecular-weight (LMW) species (i.e., 2.0-fold decreases. Specifically, APOC1 and most of the truncated products exhibited ≥1.5-fold decreases in the CRC plasma sample. The APOC1 level was previously found to be higher in preoperative serum than in postoperative serum obtained from pancreatic cancer patients;67 however, many other studies showed that the protein level was reduced in cancer serum samples, including breast cancer, papillary thyroid carcinoma, and stomach cancer.68−70 In addition, Engwegen et al. reported that the APOC1 level decreased in serum samples of CRC patients compared with those of controls and adenomatous polyps patients from their SELDI-TOF analysis.71 As for the TTR, the levels of protein and its proteoform with SAAV from Cys30Gly were found to decrease 2.0-fold, respectively, in the CRC plasma sample, whereas the S-sulfonated form exhibited a 1.5-fold increase in the CRC plasma sample. (See Figure 4D of the relative abundances of the SAA1 proteoforms.) When we summed all of the peak area values for TTR and the proteoforms and calculated the relative abundance ratio based on the peak area values, the TTR proteoforms exhibited >2.0-fold decreases in the CRC plasma sample. While TTR binds and transports the thyroid hormones and the retinol-binding protein-retinal complex,72 it has been found that different levels of TTR and the proteoforms in serum is linked to several cancers, including lung, ovarian, and endometrial cancer.73−75 It was also found that the levels of TTR and its proteoforms with S-cysteinylation and S-glutathionylation decreased in serum samples from colorectal adenoma and CRC patients,76 which correlates with our results for the TTR abundance in the CRC plasma sample. While several previous reports support our quantitative results obtained from top-down mass spectrometric analysis, we performed Western blot experiments for the six LMW proteins that showed >1.5-fold changes between the HC and CRC plasma samples to further confirm the mass spectral results. The levels of PF4, CXCL7, SAA1, CCL14, APOC1, and TTR were examined via Western blotting, and APOA1, exhibiting a 1.1-fold change between the HC and CRC plasma samples, was also included for Western blot analysis. As seen in Supporting Information Figure S13 and Figure 4E,F, the four LMW proteins (PF4, CXCL7, SAA1, and CCL14) were expressed at higher levels in the CRC plasma sample, whereas the APOC1 and TTR levels were reduced in the CRC plasma sample. The APOA1 level between the two plasma samples was also found to be similar, implying
that the Western blot results correlate with the quantitative results obtained from top-down mass spectrometric analysis. Although the Western blot results examined for the six proteins showing >1.5-fold changes between the HC and CRC plasma samples correlated with the mass spectral results, further analysis using biological replicates is needed to confirm the observed changes between the two plasma samples are significant. While we did not perform comparative analysis using the HC and CRC plasma samples with immunoaffinity depletion in the current study, we think that the current top-down platform can be applied to the comparative analysis of the plasma samples with immunoaffinity depletion; however, in the case of the plasma samples with immunoaffinity depletion, the depletion process needs to be well controlled so that the immunoaffinity depletion efficiency can be similar to the samples used for quantitative analysis. Because the top-down mass spectrometry allows quantitation of cleaved products of the higher molecular weight proteins, it is expected that the extent of immunoaffinity depletion of high-abundance proteins will also affect the quantitation of the cleaved products from the higher molecular weight proteins. In the present study, we also had to run extra LC−MS/MS runs prior to LC−MS analyses for the purpose of identification and then calculated the relative abundance ratios of the peak areas using LC−MS data to acquire more accurate quantitative information; however, it is expected that a state-ofart mass spectrometer with faster duty cycle can allow both identification and quantitation using the LC−MS/MS data.
■
CONCLUSIONS The current top-down mass spectrometric analysis of LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins allowed for identification and characterization of both LMW proteins and cleaved products of larger proteins, including those with PTMs and SAAVs. Specifically, from the depleted plasma samples, identification of cleaved products from the high-abundance proteins, which were supposed to be removed from the immunoaffinity depletion, largely decreased, facilitating identification of the LMW proteoforms derived from more different kinds of proteins, including those with lower abundances. When comparative analysis for plasma samples without removal of high-abundance proteins between HCs and CRC patients was further pursued using top-down mass spectrometry, tens of LMW proteoforms changed >1.5-fold between the HC and CRC plasma samples by calculating the ratios of peak areas from the XICs. The Western blot results examined for the six proteins were also consistent with the mass spectral results. Although the LMW proteoforms exhibiting level changes between the HC and CRC plasma samples in the current study need to be validated using a large number of biological replicates, we believe that the current top-down platform can be used to discover candidate biomarkers that show differential abundance levels or undergo PTMs and SAAVs in disease states.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00773. Figures S1−S13 include slab gel visualizations of the GELFrEE fractionation, Venn diagram showing the M
DOI: 10.1021/acs.jproteome.5b00773 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
■
(12) Greening, D. W.; Simpson, R. J. A centrifugal ultrafiltration strategy for isolating the low-molecular weight (
