- Email: [email protected]
mass spectrometry
Analytical Biochemistry 405 (2010) 132–134
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Direct monitoring of albumin lysine-525 N-homocysteinylation in human serum by liquid chromatography/mass spectrometry Marta Sikora a, Łukasz Marczak a, Tomasz Twardowski a, Maciej Stobiecki a, Hieronim Jakubowski a,b,* a b
´ , Poland Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan Department of Microbiology and Molecular Genetics, International Center for Public Health, UMDNJ–New Jersey Medical School, Newark, NJ 07101, USA
a r t i c l e
i n f o
Article history: Received 1 March 2010 Received in revised form 27 April 2010 Accepted 28 April 2010 Available online 5 July 2010 Keywords: Homocysteine-thiolactone Albumin lysine N-homocysteinylation Site-specific modification Cystathionine b-synthase deficiency LC/MS
a b s t r a c t A posttranslational protein modification by homocysteine-thiolactone (N-homocysteinylation) is linked to human vascular and neurodegenerative diseases. Although chemical and immunological methods are available to detect and quantify the extent of protein N-homocysteinylation, the determination of site-specific N-homocysteinylation in vivo remains challenging. Here we describe a liquid chromatography/mass spectrometry method that monitors the extent of N-homocysteinylation at albumin lysine-525 in vivo directly in human serum. Using this method, we found that the extent of lysine-525 N-homocysteinylation was significantly increased in patients with cystathionine b-synthase deficiency. Ó 2010 Elsevier Inc. All rights reserved.
Homocysteine (Hcy)1 and Hcy-thiolactone are intermediary metabolites that are implicated in the pathology of human cardiovascular and neurodegenerative diseases [1,2]. Hcy arises from the essential dietary protein amino acid methionine, whereas Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA (transfer RNA) synthetase [3,4] (Scheme 1). Because Hcy-thiolactone is chemically reactive, it readily modifies protein lysine amino groups to form stable isopeptide bonds (N-Hcy-protein) [3,5] (Scheme 1). This reaction alters protein structure and function [5], causes protein damage [5–8] by a thiyl radical mechanism [9], and can lead to pathological consequences such as an autoimmune response and thrombosis. For example, autoantibodies directed against N-Hcy-protein are present in humans and are elevated in stroke [10] and coronary heart disease [11] patients. Elevated thrombogenic N-Hcy-fibrinogen [12] accounts at least in part for increased atherothrombosis observed in cystathionine b-synthase (CBS)-deficient patients [13]. Furthermore, human clinical studies show that elevated plasma N-Hcy-protein levels are associated with increased risk of coronary heart disease [14], whereas
* Corresponding author at: Department of Microbiology and Molecular Genetics, International Center for Public Health, UMDNJ–New Jersey Medical School, Newark, NJ 07101, USA. Fax: +1 973 972 8981. E-mail address: [email protected] (H. Jakubowski). 1 Abbreviations used: Hcy, homocysteine; tRNA, transfer RNA; CBS, cystathionine bsynthase; HPLC, high-performance liquid chromatography; UV, ultraviolet; Lys525, lysine-525; LC/MS, liquid chromatography/mass spectrometry; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-flight; Lys137, lysine-137. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.04.034
elevated plasma Hcy-thiolactone levels are associated with the development and progression of diabetic macrovasculopathy [15]. The quantitative analysis of N-Hcy-protein levels in vivo requires acid hydrolysis, during which protein N-linked Hcy is converted to Hcy-thiolactone, which is then detected and quantified by high-performance liquid chromatography (HPLC) with ultraviolet (UV) [16] or fluorescence [17] detection. Immunological methods using rabbit polyclonal anti-N-Hcy-protein antibodies are also available for the detection of N-Hcy-protein [14,18]. Chemical tagging methods, using fluorescent or biotin aldehyde tags, for the analysis of N-Hcy-proteins are being explored [19]. Approximately 70% of circulating Hcy is N-linked to blood proteins, mostly albumin and hemoglobin (the other 30% include disulfide bound and free Hcy forms and Hcy-thiolactone) [16]. In human plasma, albumin is the major target for N-homocysteinylation by Hcy-thiolactone both in vitro [5] and in vivo [6]. Lysine-525 (Lys525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo, as shown by the identification of 525K*QTALVELVK534 peptide carrying N-linked Hcy at Lys525 (525K*) in both Hcy-thiolactone-modified and native albumin [6]. However, analyses of site-specific N-homocysteinylation in vivo require extensive sample workup and enrichment procedures. To overcome these limitations, we have developed a new liquid chromatography/mass spectrometry (LC/MS) method for monitoring the levels of albumin N-Hcy-Lys525 peptide directly in human plasma samples subjected to tryptic digestion. To detect site-specific N-homocysteinylation in vivo, we first set out to determine which lysine residues in human serum albumin
Notes & Tips / Anal. Biochem. 405 (2010) 132–134
133
Scheme 1. Metabolic conversion of Hcy to Hcy-thiolactone catalyzed by methionyl-tRNA synthetase (MetRS) (Eq. 1) and the reaction of Hcy-thiolactone with e-amino group of protein lysine residue afford posttranslationally modified protein, N-Hcy-protein (Eq. 2) [3,5].
Fig. 1. Extracted ion chromatograms of albumin peptides obtained from a tryptic digest of human serum. (A) Reference peptide 42LVNEVTEFAK51 (m/z 575.3). (B) N-Hcypeptide 137K*YLYEIAR144 (m/z 615.4). (C) N-Hcy-peptide 525K*QTALVELVK534 (m/z 651.3). K* denotes N-homocysteinylated lysine residue. (D) Normalized levels of the N-HcyLys525 peptide (m/z 651.3) are positively correlated with plasma total Hcy levels in healthy (s) and CBS-deficient (d) human subjects.
are susceptible to the modification with Hcy-thiolactone and which peptides containing N-Hcy-Lys residues can be detected. For this purpose, N-Hcy-albumin containing 6 mol of Hcy/mol albumin was prepared by an overnight incubation of human serum albumin (10 mg/ml) with 10 mM L-Hcy-thiolactoneHCl in 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 mM ethylenediaminetetraacetic acid (EDTA) at 22 °C [5]. N-Hcy-albumin (1 nmole) was then reduced with 45 mM dithiothreitol (DTT) in 0.1 M ammo-
nium bicarbonate, alkylated with 0.1 M iodoacetate, and digested with trypsin (Promega) at a trypsin/protein ratio of 1:50 at 37 °C for 17 h. Tryptic peptides were fractionated on a C18 microcolumn (ZipTip, Millipore) using 10%, 30%, 50%, and 100% acetonitrile [8] or on a C18 HPLC column (Merck). Each fraction was applied on a Prespotted AnchorChip and analyzed on a matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) Autoflex instrument (Bruker Daltonics). Peptide identification was performed by
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Mascot (http://www.matrixscience.com), allowing a mass increase of 57 Da due to carbamidomethylation and of 174 Da due to both N-homocysteinylation and carbamidomethylation. We found that K4 (1DAHK*SEVAHR10, m/z 1323.6), K12 (11FK*DLGEENFK20, m/z 1400.7), K137 (137K*YLYEIAR144, m/z 1229.7), K159 (146HPYFYAPELLFFAK*R160, m/z 2073.1), K205 (200CASLQK*FGER209, m/z 1369.6), and K212 (210AFK*AWAVAR218, m/z 1193.6), in addition to previously identified K525 (525K*QTALVELVK534, m/z 1302.8) [6] in human serum albumin, can be modified by Hcy-thiolactone in vitro. Knowing the masses of Hcy-containing peptides derived from the in vitro prepared N-Hcy-albumin, we then set out to determine whether these peptides can be detected in samples prepared from native human serum. For this purpose, human serum was diluted 60-fold with 50 mM NH4HCO3 and reduced with 1 mM DTT at 95 °C for 5 min. Thiol groups were blocked with 4 mM iodoacetamide at 22 °C in the dark for 20 min, and the serum protein was digested with sequencing-grade trypsin (trypsin/protein ratio of 1:50) at 37 °C for 17 h. Tryptic digests were subjected to LC/MS analysis using a nanoscale LC system (EASY-nLC, Proxeon) coupled to a quadrupole TOF mass spectrometer (microTOF-Q, Bruker Daltonics). Samples (10 ll) were injected onto a C18 precolumn (5 lm i.d., Nano Separations) equilibrated with 0.1% formic acid and separated on a C18 column (100 lm i.d., 150 mm, Nano Separations) using a 0–54% acetonitrile gradient in 0.1% formic acid for 30 min. Data were analyzed using Data Analysis and Bio Tools software (Bruker Daltonics). We found that the predominant 525 * K QTALVELVK534 albumin peptide was present in each analyzed serum sample, whereas other N-Hcy-peptides, because of their lower abundance, were detected in some, but not all, samples. An example of LC/MS analysis of albumin N-homocysteinylation in a human serum sample is shown in Fig. 1. To determine whether the amount of 525K*QTALVELVK534 peptide reflects the extent of total serum protein N-homocysteinylation, we analyzed levels of the m/z 651.3 peptide in tryptic digests of plasma samples from healthy individuals (prepared as described above for analyses of albumin), who have low levels of N-Hcy-protein, and from CBS-deficient patients, who have elevated levels of N-Hcy-protein [13]. For quantification purposes, signal intensity of the m/z 651.3 peptide was normalized to signal intensity of a major albumin peptide, 42LVNEVTEFAK51 (m/z 575.3). Our LC/MS assay was developed by monitoring N-Hcy-peptides obtained by trypsinolysis of in vitro modified human serum albumin. We used a broad spectrum of concentrations of albumin, and all of the parameters of the mass spectrometer were tested and set up correctly. In these experiments, all analyses were made in duplicates and standard deviations of measured peak values were 65%. All analyses of serum samples were repeated twice, and for peptides containing N-Hcy-Lys525 and N-Hcy-Lys137 (lysine-137), we observed still good intraassay variability 610%. Interassay accuracy, determined from duplicate assays of 20 human plasma samples on 2 different days, was 43%. Because the reproducibility of analyses of other N-Hcy-peptides was much worse due to their low signal/noise ratios, we did not quantify them. We found that the normalized levels of the m/z 651.3 peptide (containing N-Hcy-Lys525) were significantly higher in CBS-deficient patients (n = 15) [4,13] compared with healthy individuals (n = 29) [20] (0.0399 ± 0.0260 vs. 0.0102 ± 0.0121, P = 0.0007). These values suggest that approximately 1% and 4% albumin molecules are N-homocysteinylated at Lys525 in healthy individuals and CBS-deficient patients, respectively. The higher levels of Nhomocysteinylation at Lys525 in albumin from CBS-deficient patients reflect higher total Hcy and N-Hcy-protein levels in these pa-
tients [13] compared with healthy individuals [20], that were measured previously using chemical assays [17]. There was a significant correlation between the normalized levels of the N-HcyLys525 peptide and plasma total Hcy levels (Fig. 1D). In conclusion, the assay described in this article allows monitoring of albumin Lys525 N-homocysteinylation directly in trypsindigested human plasma. The utility of the assay was demonstrated by showing that N-homocysteinylation of Lys525 is increased in human CBS-deficient patients. Acknowledgments This work was supported in part by grants from the American Heart Association (0855919D) and the Ministry of Science and Higher Education, Poland (NN 401 230634, N401 065 32/1504, N401 132 32/2670, N204 053 32/1226, POIG.01.03.01-00-097/08). References [1] S.R. Lentz, Mechanisms of homocysteine-induced atherothrombosis, J. Thromb. Haemost. 3 (2005) 1646–1654. [2] H. Jakubowski, The molecular basis of homocysteine thiolactone-mediated vascular disease, Clin. Chem. Lab. Med. 45 (2007) 1704–1716. [3] H. Jakubowski, Metabolism of homocysteine thiolactone in human cell cultures: possible mechanism for pathological consequences of elevated homocysteine levels, J. Biol. Chem. 272 (1997) 1935–1942. [4] G. Chwatko, G.H. Boers, K.A. Strauss, D.M. Shih, H. Jakubowski, Mutations in methylenetetrahydrofolate reductase or cystathionine b-synthase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice, FASEB J. 21 (2007) 1707–1713. [5] H. Jakubowski, Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels, FASEB J. 13 (1999) 2277–2283. [6] R. Glowacki, H. Jakubowski, Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation, J. Biol. Chem. 279 (2004) 10864–10871. [7] H. Jakubowski, Molecular basis of homocysteine toxicity in humans, Cell. Mol. Life Sci. 61 (2004) 470–487. [8] J. Perla-Kajan, L. Marczak, L. Kajan, P. Skowronek, T. Twardowski, H. Jakubowski, Modification by homocysteine thiolactone affects redox status of cytochrome c, Biochemistry 46 (2007) 6225–6231. [9] M. Sibrian-Vazquez, J.O. Escobedo, S. Lim, G.K. Samoei, R.M. Strongin, Homocystamides promote free-radical and oxidative damage to proteins, Proc. Natl. Acad. Sci. USA 107 (2010) 551–554. [10] A. Undas, J. Perla, M. Lacinski, W. Trzeciak, R. Kazmierski, H. Jakubowski, Autoantibodies against N-homocysteinylated proteins in humans: implications for atherosclerosis, Stroke 35 (2004) 1299–1304. [11] A. Undas, E. Stepien, R. Glowacki, J. Tisonczyk, W. Tracz, H. Jakubowski, Folic acid administration and antibodies against homocysteinylated proteins in subjects with hyperhomocysteinemia, Thromb. Haemost. 96 (2006) 342–347. [12] D.L. Sauls, E. Lockhart, M.E. Warren, A. Lenkowski, S.E. Wilhelm, M. Hoffman, Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia, Biochemistry 45 (2006) 2480–2487. [13] H. Jakubowski, G.H. Boers, K.A. Strauss, Mutations in cystathionine b-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans, FASEB J. 22 (2008) 4071–4076. [14] X. Yang, Y. Gao, J. Zhou, Y. Zhen, Y. Yang, J. Wang, L. Song, Y. Liu, H. Xu, Z. Chen, R. Hui, Plasma homocysteine thiolactone adducts associated with risk of coronary heart disease, Clin. Chim. Acta 364 (2006) 230–234. [15] W. Gu, J. Lu, G. Yang, J. Dou, Y. Mu, J. Meng, C. Pan, Plasma homocysteine thiolactone associated with risk of macrovasculopathy in Chinese patients with type 2 diabetes mellitus, Adv. Ther. 25 (2008) 914–924. [16] H. Jakubowski, Homocysteine is a protein amino acid in humans: implications for homocysteine-linked disease, J. Biol. Chem. 277 (2002) 30425–30428. [17] H. Jakubowski, New method for the determination of protein N-linked homocysteine, Anal. Biochem. 380 (2008) 257–261. [18] J. Perla-Kajan, O. Stanger, M. Luczak, A. Ziolkowska, L.K. Malendowicz, T. Twardowski, S. Lhotak, R.C. Austin, H. Jakubowski, Immunohistochemical detection of N-homocysteinylated proteins in humans and mice, Biomed. Pharmacother. 62 (2008) 473–479. [19] T. Zang, S. Dai, D. Chen, B.W. Lee, S. Liu, B.L. Karger, Z.S. Zhou, Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes, Anal. Chem. 81 (2009) 9065–9071. [20] M. Lacinski, W. Skorupski, A. Cieslinski, J. Sokolowska, W.H. Trzeciak, H. Jakubowski, Determinants of homocysteine-thiolactonase activity of the paraoxonase-1 (PON1) protein in humans, Cell. Mol. Biol. (Noisy-le-grand) 50 (2004) 885–893.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Direct monitoring of albumin lysine-525 N-homocysteinylation in human serum by liquid chromatography/mass spectrometry Marta Sikora a, Łukasz Marczak a, Tomasz Twardowski a, Maciej Stobiecki a, Hieronim Jakubowski a,b,* a b
´ , Poland Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan Department of Microbiology and Molecular Genetics, International Center for Public Health, UMDNJ–New Jersey Medical School, Newark, NJ 07101, USA
a r t i c l e
i n f o
Article history: Received 1 March 2010 Received in revised form 27 April 2010 Accepted 28 April 2010 Available online 5 July 2010 Keywords: Homocysteine-thiolactone Albumin lysine N-homocysteinylation Site-specific modification Cystathionine b-synthase deficiency LC/MS
a b s t r a c t A posttranslational protein modification by homocysteine-thiolactone (N-homocysteinylation) is linked to human vascular and neurodegenerative diseases. Although chemical and immunological methods are available to detect and quantify the extent of protein N-homocysteinylation, the determination of site-specific N-homocysteinylation in vivo remains challenging. Here we describe a liquid chromatography/mass spectrometry method that monitors the extent of N-homocysteinylation at albumin lysine-525 in vivo directly in human serum. Using this method, we found that the extent of lysine-525 N-homocysteinylation was significantly increased in patients with cystathionine b-synthase deficiency. Ó 2010 Elsevier Inc. All rights reserved.
Homocysteine (Hcy)1 and Hcy-thiolactone are intermediary metabolites that are implicated in the pathology of human cardiovascular and neurodegenerative diseases [1,2]. Hcy arises from the essential dietary protein amino acid methionine, whereas Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA (transfer RNA) synthetase [3,4] (Scheme 1). Because Hcy-thiolactone is chemically reactive, it readily modifies protein lysine amino groups to form stable isopeptide bonds (N-Hcy-protein) [3,5] (Scheme 1). This reaction alters protein structure and function [5], causes protein damage [5–8] by a thiyl radical mechanism [9], and can lead to pathological consequences such as an autoimmune response and thrombosis. For example, autoantibodies directed against N-Hcy-protein are present in humans and are elevated in stroke [10] and coronary heart disease [11] patients. Elevated thrombogenic N-Hcy-fibrinogen [12] accounts at least in part for increased atherothrombosis observed in cystathionine b-synthase (CBS)-deficient patients [13]. Furthermore, human clinical studies show that elevated plasma N-Hcy-protein levels are associated with increased risk of coronary heart disease [14], whereas
* Corresponding author at: Department of Microbiology and Molecular Genetics, International Center for Public Health, UMDNJ–New Jersey Medical School, Newark, NJ 07101, USA. Fax: +1 973 972 8981. E-mail address: [email protected] (H. Jakubowski). 1 Abbreviations used: Hcy, homocysteine; tRNA, transfer RNA; CBS, cystathionine bsynthase; HPLC, high-performance liquid chromatography; UV, ultraviolet; Lys525, lysine-525; LC/MS, liquid chromatography/mass spectrometry; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; MALDI–TOF, matrix-assisted laser desorption/ionization time-of-flight; Lys137, lysine-137. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.04.034
elevated plasma Hcy-thiolactone levels are associated with the development and progression of diabetic macrovasculopathy [15]. The quantitative analysis of N-Hcy-protein levels in vivo requires acid hydrolysis, during which protein N-linked Hcy is converted to Hcy-thiolactone, which is then detected and quantified by high-performance liquid chromatography (HPLC) with ultraviolet (UV) [16] or fluorescence [17] detection. Immunological methods using rabbit polyclonal anti-N-Hcy-protein antibodies are also available for the detection of N-Hcy-protein [14,18]. Chemical tagging methods, using fluorescent or biotin aldehyde tags, for the analysis of N-Hcy-proteins are being explored [19]. Approximately 70% of circulating Hcy is N-linked to blood proteins, mostly albumin and hemoglobin (the other 30% include disulfide bound and free Hcy forms and Hcy-thiolactone) [16]. In human plasma, albumin is the major target for N-homocysteinylation by Hcy-thiolactone both in vitro [5] and in vivo [6]. Lysine-525 (Lys525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo, as shown by the identification of 525K*QTALVELVK534 peptide carrying N-linked Hcy at Lys525 (525K*) in both Hcy-thiolactone-modified and native albumin [6]. However, analyses of site-specific N-homocysteinylation in vivo require extensive sample workup and enrichment procedures. To overcome these limitations, we have developed a new liquid chromatography/mass spectrometry (LC/MS) method for monitoring the levels of albumin N-Hcy-Lys525 peptide directly in human plasma samples subjected to tryptic digestion. To detect site-specific N-homocysteinylation in vivo, we first set out to determine which lysine residues in human serum albumin
Notes & Tips / Anal. Biochem. 405 (2010) 132–134
133
Scheme 1. Metabolic conversion of Hcy to Hcy-thiolactone catalyzed by methionyl-tRNA synthetase (MetRS) (Eq. 1) and the reaction of Hcy-thiolactone with e-amino group of protein lysine residue afford posttranslationally modified protein, N-Hcy-protein (Eq. 2) [3,5].
Fig. 1. Extracted ion chromatograms of albumin peptides obtained from a tryptic digest of human serum. (A) Reference peptide 42LVNEVTEFAK51 (m/z 575.3). (B) N-Hcypeptide 137K*YLYEIAR144 (m/z 615.4). (C) N-Hcy-peptide 525K*QTALVELVK534 (m/z 651.3). K* denotes N-homocysteinylated lysine residue. (D) Normalized levels of the N-HcyLys525 peptide (m/z 651.3) are positively correlated with plasma total Hcy levels in healthy (s) and CBS-deficient (d) human subjects.
are susceptible to the modification with Hcy-thiolactone and which peptides containing N-Hcy-Lys residues can be detected. For this purpose, N-Hcy-albumin containing 6 mol of Hcy/mol albumin was prepared by an overnight incubation of human serum albumin (10 mg/ml) with 10 mM L-Hcy-thiolactoneHCl in 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 mM ethylenediaminetetraacetic acid (EDTA) at 22 °C [5]. N-Hcy-albumin (1 nmole) was then reduced with 45 mM dithiothreitol (DTT) in 0.1 M ammo-
nium bicarbonate, alkylated with 0.1 M iodoacetate, and digested with trypsin (Promega) at a trypsin/protein ratio of 1:50 at 37 °C for 17 h. Tryptic peptides were fractionated on a C18 microcolumn (ZipTip, Millipore) using 10%, 30%, 50%, and 100% acetonitrile [8] or on a C18 HPLC column (Merck). Each fraction was applied on a Prespotted AnchorChip and analyzed on a matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) Autoflex instrument (Bruker Daltonics). Peptide identification was performed by
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Notes & Tips / Anal. Biochem. 405 (2010) 132–134
Mascot (http://www.matrixscience.com), allowing a mass increase of 57 Da due to carbamidomethylation and of 174 Da due to both N-homocysteinylation and carbamidomethylation. We found that K4 (1DAHK*SEVAHR10, m/z 1323.6), K12 (11FK*DLGEENFK20, m/z 1400.7), K137 (137K*YLYEIAR144, m/z 1229.7), K159 (146HPYFYAPELLFFAK*R160, m/z 2073.1), K205 (200CASLQK*FGER209, m/z 1369.6), and K212 (210AFK*AWAVAR218, m/z 1193.6), in addition to previously identified K525 (525K*QTALVELVK534, m/z 1302.8) [6] in human serum albumin, can be modified by Hcy-thiolactone in vitro. Knowing the masses of Hcy-containing peptides derived from the in vitro prepared N-Hcy-albumin, we then set out to determine whether these peptides can be detected in samples prepared from native human serum. For this purpose, human serum was diluted 60-fold with 50 mM NH4HCO3 and reduced with 1 mM DTT at 95 °C for 5 min. Thiol groups were blocked with 4 mM iodoacetamide at 22 °C in the dark for 20 min, and the serum protein was digested with sequencing-grade trypsin (trypsin/protein ratio of 1:50) at 37 °C for 17 h. Tryptic digests were subjected to LC/MS analysis using a nanoscale LC system (EASY-nLC, Proxeon) coupled to a quadrupole TOF mass spectrometer (microTOF-Q, Bruker Daltonics). Samples (10 ll) were injected onto a C18 precolumn (5 lm i.d., Nano Separations) equilibrated with 0.1% formic acid and separated on a C18 column (100 lm i.d., 150 mm, Nano Separations) using a 0–54% acetonitrile gradient in 0.1% formic acid for 30 min. Data were analyzed using Data Analysis and Bio Tools software (Bruker Daltonics). We found that the predominant 525 * K QTALVELVK534 albumin peptide was present in each analyzed serum sample, whereas other N-Hcy-peptides, because of their lower abundance, were detected in some, but not all, samples. An example of LC/MS analysis of albumin N-homocysteinylation in a human serum sample is shown in Fig. 1. To determine whether the amount of 525K*QTALVELVK534 peptide reflects the extent of total serum protein N-homocysteinylation, we analyzed levels of the m/z 651.3 peptide in tryptic digests of plasma samples from healthy individuals (prepared as described above for analyses of albumin), who have low levels of N-Hcy-protein, and from CBS-deficient patients, who have elevated levels of N-Hcy-protein [13]. For quantification purposes, signal intensity of the m/z 651.3 peptide was normalized to signal intensity of a major albumin peptide, 42LVNEVTEFAK51 (m/z 575.3). Our LC/MS assay was developed by monitoring N-Hcy-peptides obtained by trypsinolysis of in vitro modified human serum albumin. We used a broad spectrum of concentrations of albumin, and all of the parameters of the mass spectrometer were tested and set up correctly. In these experiments, all analyses were made in duplicates and standard deviations of measured peak values were 65%. All analyses of serum samples were repeated twice, and for peptides containing N-Hcy-Lys525 and N-Hcy-Lys137 (lysine-137), we observed still good intraassay variability 610%. Interassay accuracy, determined from duplicate assays of 20 human plasma samples on 2 different days, was 43%. Because the reproducibility of analyses of other N-Hcy-peptides was much worse due to their low signal/noise ratios, we did not quantify them. We found that the normalized levels of the m/z 651.3 peptide (containing N-Hcy-Lys525) were significantly higher in CBS-deficient patients (n = 15) [4,13] compared with healthy individuals (n = 29) [20] (0.0399 ± 0.0260 vs. 0.0102 ± 0.0121, P = 0.0007). These values suggest that approximately 1% and 4% albumin molecules are N-homocysteinylated at Lys525 in healthy individuals and CBS-deficient patients, respectively. The higher levels of Nhomocysteinylation at Lys525 in albumin from CBS-deficient patients reflect higher total Hcy and N-Hcy-protein levels in these pa-
tients [13] compared with healthy individuals [20], that were measured previously using chemical assays [17]. There was a significant correlation between the normalized levels of the N-HcyLys525 peptide and plasma total Hcy levels (Fig. 1D). In conclusion, the assay described in this article allows monitoring of albumin Lys525 N-homocysteinylation directly in trypsindigested human plasma. The utility of the assay was demonstrated by showing that N-homocysteinylation of Lys525 is increased in human CBS-deficient patients. Acknowledgments This work was supported in part by grants from the American Heart Association (0855919D) and the Ministry of Science and Higher Education, Poland (NN 401 230634, N401 065 32/1504, N401 132 32/2670, N204 053 32/1226, POIG.01.03.01-00-097/08). References [1] S.R. Lentz, Mechanisms of homocysteine-induced atherothrombosis, J. Thromb. Haemost. 3 (2005) 1646–1654. [2] H. Jakubowski, The molecular basis of homocysteine thiolactone-mediated vascular disease, Clin. Chem. Lab. Med. 45 (2007) 1704–1716. [3] H. Jakubowski, Metabolism of homocysteine thiolactone in human cell cultures: possible mechanism for pathological consequences of elevated homocysteine levels, J. Biol. Chem. 272 (1997) 1935–1942. [4] G. Chwatko, G.H. Boers, K.A. Strauss, D.M. Shih, H. Jakubowski, Mutations in methylenetetrahydrofolate reductase or cystathionine b-synthase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice, FASEB J. 21 (2007) 1707–1713. [5] H. Jakubowski, Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels, FASEB J. 13 (1999) 2277–2283. [6] R. Glowacki, H. Jakubowski, Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation, J. Biol. Chem. 279 (2004) 10864–10871. [7] H. Jakubowski, Molecular basis of homocysteine toxicity in humans, Cell. Mol. Life Sci. 61 (2004) 470–487. [8] J. Perla-Kajan, L. Marczak, L. Kajan, P. Skowronek, T. Twardowski, H. Jakubowski, Modification by homocysteine thiolactone affects redox status of cytochrome c, Biochemistry 46 (2007) 6225–6231. [9] M. Sibrian-Vazquez, J.O. Escobedo, S. Lim, G.K. Samoei, R.M. Strongin, Homocystamides promote free-radical and oxidative damage to proteins, Proc. Natl. Acad. Sci. USA 107 (2010) 551–554. [10] A. Undas, J. Perla, M. Lacinski, W. Trzeciak, R. Kazmierski, H. Jakubowski, Autoantibodies against N-homocysteinylated proteins in humans: implications for atherosclerosis, Stroke 35 (2004) 1299–1304. [11] A. Undas, E. Stepien, R. Glowacki, J. Tisonczyk, W. Tracz, H. Jakubowski, Folic acid administration and antibodies against homocysteinylated proteins in subjects with hyperhomocysteinemia, Thromb. Haemost. 96 (2006) 342–347. [12] D.L. Sauls, E. Lockhart, M.E. Warren, A. Lenkowski, S.E. Wilhelm, M. Hoffman, Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia, Biochemistry 45 (2006) 2480–2487. [13] H. Jakubowski, G.H. Boers, K.A. Strauss, Mutations in cystathionine b-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans, FASEB J. 22 (2008) 4071–4076. [14] X. Yang, Y. Gao, J. Zhou, Y. Zhen, Y. Yang, J. Wang, L. Song, Y. Liu, H. Xu, Z. Chen, R. Hui, Plasma homocysteine thiolactone adducts associated with risk of coronary heart disease, Clin. Chim. Acta 364 (2006) 230–234. [15] W. Gu, J. Lu, G. Yang, J. Dou, Y. Mu, J. Meng, C. Pan, Plasma homocysteine thiolactone associated with risk of macrovasculopathy in Chinese patients with type 2 diabetes mellitus, Adv. Ther. 25 (2008) 914–924. [16] H. Jakubowski, Homocysteine is a protein amino acid in humans: implications for homocysteine-linked disease, J. Biol. Chem. 277 (2002) 30425–30428. [17] H. Jakubowski, New method for the determination of protein N-linked homocysteine, Anal. Biochem. 380 (2008) 257–261. [18] J. Perla-Kajan, O. Stanger, M. Luczak, A. Ziolkowska, L.K. Malendowicz, T. Twardowski, S. Lhotak, R.C. Austin, H. Jakubowski, Immunohistochemical detection of N-homocysteinylated proteins in humans and mice, Biomed. Pharmacother. 62 (2008) 473–479. [19] T. Zang, S. Dai, D. Chen, B.W. Lee, S. Liu, B.L. Karger, Z.S. Zhou, Chemical methods for the detection of protein N-homocysteinylation via selective reactions with aldehydes, Anal. Chem. 81 (2009) 9065–9071. [20] M. Lacinski, W. Skorupski, A. Cieslinski, J. Sokolowska, W.H. Trzeciak, H. Jakubowski, Determinants of homocysteine-thiolactonase activity of the paraoxonase-1 (PON1) protein in humans, Cell. Mol. Biol. (Noisy-le-grand) 50 (2004) 885–893.