- Email: [email protected]
Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry
Journal Pre-proof Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing Mass spectrometry
Cai Tie, Lili Liu, Teng Feng, Rina Sa, Qiangwei Xia, Handong Liang, Yonghui Mao PII:
S1874-3919(20)30044-0
DOI:
https://doi.org/10.1016/j.jprot.2020.103676
Reference:
JPROT 103676
To appear in:
Journal of Proteomics
Received date:
10 December 2019
Revised date:
10 January 2020
Accepted date:
28 January 2020
Please cite this article as: C. Tie, L. Liu, T. Feng, et al., Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry, Journal of Proteomics (2019), https://doi.org/10.1016/ j.jprot.2020.103676
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Differential
Analysis
of
Urinary
Albumin
for Membranous
Nephropathy Patients by Online Capillary Isoelectric Focusing - Mass Spectrometry Cai Tie*‡1,2, Lili Liu‡ 3, Teng Feng4, Rina Sa4, Qiangwei Xia4,5, Handong Liang1, Yonghui Mao **3 1
State Key Laboratory of Coal Resources and Safe Mining, China University of Mining and Technology
(Beijing), Beijing 100083, P.R. China 2
School of Chemical and Environmental Engineering, China University of Mining and Technology
3
of
(Beijing), Beijing 100083, P.R. China Department of Nephrology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric
ro
Medicine, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
CMP Scientific Corp, 760 Parkside Avenue, STE 211, Brooklyn, New York 11226, United States
5
EverGauge Science and Technology, ShenZhen, P.R. China
-p
4
Jo ur
na
lP
re
ABSTRACT: Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs. On the other hand, urinary albumin assay is one of the de facto tests for kidney function testing for several decades. In this work, urinary albumin species between primary and secondary MN patients are compared using a newly developed capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The distinct patterns of cationic and acidic urinary albumin species, as revealed by this novel CIEF-MS technology, suggest potential applications of this differential analysis for subtyping of membranous nephropathy. Further investigation of these cationic human albumin species in urine may provide clues to the disease onset and development of MN, thus facilitating treatment. In addition, this novel workflow of using CIEF-MS for urinary protein analysis may be beneficial to the research, pathology, prognosis, and diagnosis of many other types of kidney diseases, such as chronic kidney disease, diabetic nephrology, etc. KEY WORDS Membranous Nephropathy, capillary isoelectric focusing – mass spectrometry, Urinary Protein, Albumin
Background Membranous nephropathy （MN）is the leading cause of nephrotic syndrome in adults, affecting ~ 5–10 patients per million population[1]. Although in most patients this disease progresses relatively slowly, about
Journal Pre-proof one-third of the MN patients with nephrotic syndrome eventually develop end-stage renal diseases[2]. With regard to pathology, MN has been considered as one type of immune system complication-related diseases. Primary MN is one type of autoimmune diseases which is caused by in situ binding of circulating antibodies to an endogenous podocytic antigen, leading to the development of subepithelial immune deposits. Since the discovery of M-type phospholipase A2 receptor (PLA2R) as the first major target autoantigen, there is an increasing amount of research being put on the discovery of other antigens for primary MN diseases[3, 4]. A number of recent literatures show that, the levels of circulating anti-PLA2R antibodies play critical roles in the diagnosis and prognosis of primary MN[5]. Deposit these progresses, there are remaining questions that need to be addressed on. First, the mechanism of MN occurring is not thoroughly understood. The role of PLA2R in disease development is an ongoing research area. Besides PLA2R, there are several other novel antigens that have been recently discovered, indicating the underlying complexity of MN diseases, as well as suggesting the possible existence of many subtypes of MN diseases. To this end, Hanna
of
et al. identified cationic bovine serum albumin (cBSA) as one of the MN antigens by immunoassay study. Further studies showed that anti-cBSA antibodies were found in 2% of the MN patients[6]. These patients’
ro
diseases development and prognosis could be identified among groups of patients with other subtypes. It was proposed that, anti-cBSA antibodies bind these cBSA molecules at the anionic glomerular capillary
-p
wall, including the formation of immune complexes. However, there are missing steps in the proposed progression. In particular, there is lack of explanation for the production of anti-cBSA antibodies. Due to
re
limited evidence so far, these newly discovered biomarkers have yet to be used by physicians for diagnosis. In contrast, there are currently at least 30% of the MN patients being left with absence of PLA2R
lP
antibodies[3].
Second, although PLA2R has been an effective biomarker for MN diagnosis, it remains challenging to use it for disease subtyping. Even for primary and secondary MN patients, there is a lack of specificity in the
na
PLA2R testing. As a result, subtyping of MN patients are, at this point, mainly based on clinical experiences. Due to inaccurate and often subjective disease subtyping, the treatment of MN is not straightforward, affecting the effectiveness of the treatment. Meanwhile, recent research of rituximab therapy, which is
Jo ur
regarded as the most efficient clinical treatment for MN patients, reports independence of baseline proteinuria, pharmacokinetic studies and evaluation of B and T cell subsets[7, 8]. The identification of antiPLA2R antibodies alone is not sufficient to predict the prognosis of the MN patients. Elevated level of albumin in urine samples has been used as one of the widely accepted clinical assays for MN diseases, as well as for many other forms of kidney diseases, for decades[9]. It is well known that, when MN occurs and develops, albumin often leaks into urine and becomes the major protein component in the patient’s urine. Therefore, human serum albumin (HSA) abundance in the urine samples has been widely used as one of the metrics for MNs diagnosis and therapy efficacy monitoring. We report a direct comparison of urinary HSA composition, including the relative abundance of each HSA species, between primary and secondary MN patients, using both reversed-phase liquid chromatography – mass spectrometry (LC-MS) technology and a novel online capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The novel CIEF-MS workflow is implemented using an electrokinetically pumped sheath-flow nanospray capillary electrophoresis – mass spectrometry (CE-MS) coupling technology. With this powerful CIEF-MS method, the albumin species in each patient’s urine sample were separated according to their isoelectric point (pI) values, then introduced into high-resolution time-of-flight mass spectrometry for subsequent identification of each species by intact masses. This novel CIEF-MS approach provides two-dimensional information regarding the albumin species in patient’s urine: the pI distribution of the albumin species, as well as the molecular weight of each separated albumin species. In addition, this CIEF-MS technology allows for the separation and identification of minute
Journal Pre-proof urinary albumin variants among patients, whereas the regular LC-MS approach does not offer such separation power. With the successful application of this CIEF-MS workflow for urinary albumin analysis, we report the separation, identification and mass assignment of multiple basic and acidic HSA species from secondary MN patient’s urine sample. In contrast, the primary MN patient’s urine sample does not contain these albumin charge variant species. This study is a proof-of-concept demonstration of the potential application of the newly developed electrokinetically pumped nanospray CE-MS technology for direct human urine analysis. Method Materials. The carrier ampholytes, Pharmalyte® with pH ranges of 3.0−8.0 and 3.0−10.0, were purchased from GE Healthcare Life Sciences (USP, Pittsburgh, PA). LC-MS grade reagents, including water, formic acid, ammonium hydroxide, acetonitrile, and methanol were obtained from Millipore Sigma (Burlington, MA). The electrospray emitters for online cIEF-MS analysis (1.0 mm o.d., 0.75 mm i.d., 15−25 μm tip size) and
of
neutral coating PS2 separation capillaries (360 μm o.d., 50 μm i.d.) were supplied by CMP Scientific Corp. The CR3520 CIEF-MS reagent kit is provided by CMP Scientific Corp.
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The urine samples used for this study were collected at Beijing Hospital. The ethical policy of Beijing Hospital was followed for the procedure.
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Urine sample pretreatment. 400 µL of each urine sample was desalted using the Millipore 10K filtering device by 15 min, 14,000 rpm centrifugation, twice at room temperature.
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UPLC-HRMS albumin analysis. A Waters G2-Si Q-TOF HRMS (Waters, Massachusetts, USA) and BEH C4 reversed-phase protein separation was used for the LC-MS study. The flow rate was 40 µL/min.
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Separation was carried out with the following gradient: 15% acetonitrile aqueous solution, hold for 1min; acetonitrile concentration was then risen to 75% within 10 min; after that, the acetonitrile concentration was further increased to 85% in 5 min; the mobile phase was then brought back to initial condition within 4 min.
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The mass spectrometry source temperature was set at 150 ℃. ESI voltage was set at 4.0 kV. Mass range for data acquisition was 300 - 2,000 m/z.
Online capillary isoelectric focusing-mass spectrometry (CIEF-MS). An EMASS-II CE-MS ion source
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(CMP Scientific Corp, Brooklyn, NY, USA) was used to couple the Agilent 7100 capillary electrophoresis with the Agilent 6230 TOF mass spectrometer. Separation capillaries with a length of 75 cm were used throughout the study, in order to balance between analysis time, albumin charge variants resolution, and sensitivity. The Buffer B in the CR3520 CIEF-MS kit (CMP Scientific Corp) was used as the catholyte. The anolyte was Buffer A in the CR3520 CIEF-MS kit. The sheath liquid was the Buffer SL in the CR3520 CIEF-MS kit. Protein samples (0.1−1.0 mg/mL) were prepared in 1.5% Pharmalyte® 3-10 with an equal volume mixing with the Sample Buffer S35 in the CR3520 kit. The catholyte solution was injected under 950 mbar for 20 s, which was followed by sample injection under 950 mbar for 45 s. After catholyte and sample injection, a voltage was applied under normal polarity with a field strength of 250 V/cm. A small pressure of 5-35 mbar was applied at the capillary inlet (anode) to assist mobilization. The electrospray ionization voltage was set at 2.4 kV using the external high voltage power supply that came with the EMASS-II ion source. The distance from the emitter tip to the mass spectrometer was adjusted to 4 mm with the help of a microscope camera. MS Parameters. The capillary voltage of the TOF was set at 0 V. Drying gas was 2 L/min at 350 °C. Fragmentor voltage was 200 V. Acquisition range was m/z 700−4,500. Acquisition rate was 1 spectrum per second. The CE-MS method setup, data acquisition, and analysis were carried out using Agilent MassHunter Workstation software (v B.10).
Journal Pre-proof
Results and discussion Sources of the urine samples. As shown in Fig.1, based on biopsy results, both patients under investigation were PLA2R antibody-positive cases. Their total urine protein concentrations were measured to be 2.119 g/L and 2.570 g/L, respectively. However, the clinical presentation of these two patients were vastly different. The 24h total urinary protein of the Patient A was 1.90 g (urine volume 900 mL). The Patient B had a 24-h total urinary protein of 9.77 g (urine volume 3,800 mL). The urinary samples before buffer exchange have protein concentrations of 2.11 and 2.57 mg/mL, for Patient A and Patient B, respectively. While the Patient A was diagnosed as MN secondary to hypereosinophilia syndrome, this urine protein abundance alone is not uncommon, given that the total urinary protein assay provides limited information about disease subtyping and progression in MN. Based on combined clinical testing results, these two
of
patients received different treatments. The Patient A received glucocorticoids therapy alone, and PLA2R antibody transformed negative within a month. The Patient B received tacrolimus therapy and achieved partial remission and significant decrease of PLA2R antibody in the sixth month. Details of these two
ro
patients are listed in Table 1.
Patient A
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Table 1. Clinical diagnosis information of the two MN patients under investigation Patient B
76
Gender
Female
Male
MN subtypes
Secondary MN
Primary MN
PLA2R (RU/mL)
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Urine protein (g/24 h)
90
131
1.9
9.77
na
Creatinine (µmol/L)
54
re
Age (years)
265.82
167.98
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LC-MS albumin analysis. Urinary proteins from the two patients were analyzed by LC-MS. As shown in Fig.1, albumin was identified as the major protein component in these urine samples. Although the albumin species of both two patients’ urine were separated into two peaks, as shown in the LC-MS data, there was little difference between peak 1 and peak 2, based on their mass spectra. The albumin species in these two urine samples showed differences in their molecular weights. In the urine sample of the Patient A, the albumin species are identified as a single proteoform. However, in the urine sample of the Patient B, there were four distinct albumin species, with deconvoluted masses of 66,436 Da, 66,557 Da, 66,659 Da, and 66,718 Da. The difference in albumin species in the urine samples may be associated with MN subtypes.
Journal Pre-proof Fig.1 Averaged mass spectra of urine albumin species identified by LC-MS. A, mass spectrum of albumin in the urine sample of the Patient A; B, mass spectrum of albumin in the urine sample of the Patient B. The insets are zoom views of the mass spectral peaks, demonstrating the distinct features of albumin species in each patient’s urine sample. CIEF-MS separation condition optimization. In addition to the LC-MS analysis, a recently developed online capillary isoelectric focusing – mass spectrometry technology, CIEF-MS, was applied for further analysis of the urinary albumin species. This online CIEF-MS technology, which is based on a novel electrokinetically pumped sheath-flow nanospray CE-MS coupling technology, is a powerful protein separation and detection system for disclosing protein charge variants[10-13]. In order to facilitate albumin analysis in urine samples, a number of experimental conditions of this CIEF-MS workflow are taken from previous system optimization studies. The effects of some of the critical CIEF-MS parameters, including the duration of catholyte and sample injection, system pressure at the capillary inlet, sample concentration,
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etc., were investigated in previous studies[14]. According to previous findings, the pressure applied at the capillary inlet during the focusing and mobilization process may affect the CIEF-MS resolution and
ro
sensitivity in many ways. Figure 2 shows the comparison of various pressure levels at the capillary inlet. When 50 mbar was applied, the analysis was completed within 20 min. Whereas, under the 15 mbar
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condition, the entire analysis took more than 50 min. However, fast analysis was not without the cost of compromised performance. For example, while the basic albumin peak was easily distinguished from main
re
peak in the injection with 15 mbar running pressure, this basic albumin peak coeluted with the main peak in the analysis when 50 mbar of running pressure was applied. An optimized system pressure of 15 mbar
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na
lP
was then used throughout this study.
Fig.2 Total ion electropherograms of the CIEF-MS results with various running pressures being applied at the capillary inlet: 15 mbar (Panel A), 25 mbar (Panel B), and 50 mbar (Panel C). Urine sample analysis with CIEF-MS. Proteins isolated from the urine samples were analyzed with the above optimized CIEF-MS conditions. The major protein component was detected to be albumin species in both patients’ urine samples (Fig. 3). There are clear differences on albumin charge heterogeneity patterns between these urine samples. The urinary albumin from the Patient A is present in three charge forms: one basic peak, a main peak, and an acidic peak. As a comparison, only a main albumin peak was detected in the urine sample of the Patient B.
Journal Pre-proof Fig.3 Online CIEF-MS results of the urinary proteins from MN patients. A, results of the urine sample from the Patient A; B, results of the urine sample from the Patient B. Figure 4 shows the averaged mass spectra of each identified albumin species. The three albumin charge variants in the urine sample from the Patient A showed similar m/z mass spectra, with deconvoluted albumin masses around 66,555 Da. The albumin peak in the urine sample from the Patient B (Fig. 4D) showed a deconvoluted mass of 66,560 Da, which matching the LC-MS data quite well. Besides the main species, there are three additional albumin species as identified by unique deconvoluted masses: 66,443 Da, 66,651 Da, and 66,719 Da （Figure SI). These observations may suggest the existence of protein misfolding and post-translational modifications that are associated with the pathology of primary and secondary
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membranous nephropathy.
Fig.4 Averaged mass spectra of the CIEF-MS identified albumin species in the urine samples. Panels A, B, and C:
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averaged mass spectra of the basic, main, and acidic peaks of the albumin charge variants in the urine sample from the Patient A. Panel D: averaged mass spectra of the albumin peak in the urine sample from the Patient B. The insets
Conclusions
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show the deconvoluted masses of each identified albumin species.
Membranous nephropathy is a complex disease and may occur in many forms of subtypes, which come with
Jo ur
different pathology thus necessitating differential therapy. There is a rise in the demand for membranous nephropathy subtyping at the molecular level. Although anti-PLA2R used to be a sensitive and specific biomarker of primary MN, it may also be tested positive in the cases of secondary membranous nephropathy. As for the patient A reported above, anti-PLA2R rapidly conversed negative after glucocorticoids monotherapy, to which the therapy for primary MN will not react. We made a definite diagnosis for the case of secondary MN. In this case, diagnostic specificity of anti-PLA2R in differentiating primary MN from secondary forms was poor, suggesting an urgent need for new biomarker discovery. We report the analysis of urinary proteins from primary and secondary membranous nephropathy patients, using both regular LC-MS workflow, and a novel online CIEF-MS technology, which was developed by the authors. Compared with LC-MS method, the CIEF-MS approach that we developed showed higher resolution in terms of separating out various urinary albumin species based on their isoelectric points. In this novel CIEFMS-based urinary protein analysis workflow, both molecular weights and protein charge heterogeneity distribution information can be obtained in a fully automated manner. Both cationic and anionic human serum albumin species were identified in the urine sample of a secondary membranous nephropathy patient. This has not been previously reported in the literatures. Besides the uncovering of albumin charge variants in the secondary membranous nephropathy patient’s urine samples, clear difference on the urinary albumin species were discovered between primary and secondary MN patients by both CIEF-MS and LC-MS analysis. The differences in albumin compositions could be associated with diseases pathology. One hypothesis would be
Journal Pre-proof that, the urinary albumin may result from protein misfolding and post-translational modifications, which are modulated by membranous nephropathy disease onset and subsequent progression and development. The accurate subtyping of membranous nephropathy patients remains a challenge, even though there has been a great deal of breakthroughs, such as the discovery of PLA2R antibody. This novel CIEF-MS workflow of urinary albumin charge heterogeneity characterization, as demonstrated by this case study, may lead to a powerful diagnosis tool for distinguishing between primary and secondary membranous nephropathy patients, thus facilitating the decision-making process for physicians. To this end, future investigation which involves large cohort study of primary and secondary membranous nephropathy patients is going to provide valuable information, with regard to the effectiveness of applying this novel CIEF-MS-based urinary albumin charge heterogeneity assay, for the triage of membranous nephropathy patients.
AUTHOR INFORMATION
of
Corresponding Author
Fax: +86 10 62331029; E-mail: [email protected]
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**Corresponding author. Tel.: +86 10 85133880;
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*Corresponding author. Tel.: +86 10 62331029;
Fax: +86 10 85133880; E-mail: [email protected]
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Author Contributions ‡These authors contributed equally.
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ACKNOWLEDGMENTS
This work was supported by a grant from Beijing Hospital Nova Project (BJ-2018-131), the National Natural Science Foundation of China (no.81200523), open fund of the state key laboratory of coal resources and safe mining
Foundation (6184049).
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REFERENCES
na
(SKLCRSM19KFA20), Beijing Hospital Clinical Research 121 Project (121-2016008), and Beijing Natural Science
[1] P. Ruggenenti, F.C. Fervenza, G. Remuzzi, Treatment of membranous nephropathy: time for a paradigm shift, Nature Reviews Nephrology
(2017).
[2] J.M. Francis, L.H. Beck, D.J. Salant, Membranous Nephropathy: A Journey From Bench to Bedside, American Journal of Kidney Diseases
(2016) S0272638616007009.
[3] L.H. Beck, R.G.B. Bonegio, G. Lambeau, D.M. Beck, D.J. Salant, M-Type Phospholipase A 2 Receptor as Target Antigen in Idiopathic Membranous Nephropathy, New England Journal of Medicine 361(1) (2009) 11-21.
Journal Pre-proof [4] N.M. Tomas, L.H. Beck, C. Meyer-Schwesinger, B. Seitz-Polski, H. Ma, G. Zahner, G. Dolla, E. Hoxha, U. Helmchen, A.-S. Dabert-Gay, Thrombospondin Type-1 Domain-Containing 7A in Idiopathic Membranous Nephropathy, New England Journal of Medicine 371(24) (2014) 22772287. [5] P. Ronco, H. Debiec, Pathophysiological advances in membranous nephropathy: time for a shift in patient's care, Lancet 385(9981) (2015) 1983-1992.
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[6] H. Debiec, F. Lefeu, M.J. Kemper, P. Niaudet, G. Deschênes, G. Remuzzi, T. Ulinski, P.
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Ronco, Early-Childhood Membranous Nephropathy Due to Cationic Bovine Serum Albumin,
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New England Journal of Medicine 364(22) (2011) 2101-2110.
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[7] F.C. Fervenza, R.S. Abraham, S.B. Erickson, M.V. Irazabal, A. Eirin, U. Specks, P.H.
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D.C. Cattran, Rituximab Therapy in Idiopathic Membranous Nephropathy: A 2-Year Study, Clinical Journal of the American Society of Nephrology 5(12) (2010) 2188-2198.
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[8] D. Roccatello, S. Sciascia, D. Di Simone, L. Solfietti, C. Naretto, R. Fenoglio, S. Baldovino, E. Menegatti, New insights into immune mechanisms underlying response to Rituximab in patients with membranous nephropathy: A prospective study and a review of the literature, Autoimmunity Reviews 15(6) (2016) 529-538. [9] KDIGO, KDIGO clinical practice guidelines for glomerulonephritis, Kidney Int. Suppl. 2 (2012). [10] C.C. Liu, J. Zhang, N.J. Dovichi, A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry, Rapid Communications in Mass Spectrometry 19(2) (2005) 187-192.
Journal Pre-proof [11] J. Dai, Y. Zhang, A Middle-Up Approach with Online Capillary Isoelectric Focusing/Mass Spectrometry for In-Depth Characterization of Cetuximab Charge Heterogeneity, Analytical Chemistry 90(24) (2018) 14527-14534. [12] J. Dai, J. Lamp, Q. Xia, Y. Zhang, Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants, Anal Chem 90(3) (2018) 2246-2254.
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[13] P.W. Lindenburg, R. Haselberg, G. Rozing, R. Ramautar, Developments in Interfacing
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[14] J. Xia, R. Wang, Identification of NISTmAb Charge Variants by Fully Automated Capillary Retrieved
from
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website:
(2019).
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5994-1079en-agilent.pdf
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https://www.agilent.com/cs/library/applications/application-nistmab-charge-variants-cief-ms-
Figure 1
Figure 2
Figure 3
Figure 4
Cai Tie, Lili Liu, Teng Feng, Rina Sa, Qiangwei Xia, Handong Liang, Yonghui Mao PII:
S1874-3919(20)30044-0
DOI:
https://doi.org/10.1016/j.jprot.2020.103676
Reference:
JPROT 103676
To appear in:
Journal of Proteomics
Received date:
10 December 2019
Revised date:
10 January 2020
Accepted date:
28 January 2020
Please cite this article as: C. Tie, L. Liu, T. Feng, et al., Differential analysis of urinary albumin for membranous nephropathy patients by online capillary isoelectric focusing - Mass spectrometry, Journal of Proteomics (2019), https://doi.org/10.1016/ j.jprot.2020.103676
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Differential
Analysis
of
Urinary
Albumin
for Membranous
Nephropathy Patients by Online Capillary Isoelectric Focusing - Mass Spectrometry Cai Tie*‡1,2, Lili Liu‡ 3, Teng Feng4, Rina Sa4, Qiangwei Xia4,5, Handong Liang1, Yonghui Mao **3 1
State Key Laboratory of Coal Resources and Safe Mining, China University of Mining and Technology
(Beijing), Beijing 100083, P.R. China 2
School of Chemical and Environmental Engineering, China University of Mining and Technology
3
of
(Beijing), Beijing 100083, P.R. China Department of Nephrology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric
ro
Medicine, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China
CMP Scientific Corp, 760 Parkside Avenue, STE 211, Brooklyn, New York 11226, United States
5
EverGauge Science and Technology, ShenZhen, P.R. China
-p
4
Jo ur
na
lP
re
ABSTRACT: Membranous nephropathy (MN) is one of the most common causes of primary glomerular diseases worldwide. The M-type phospholipase A2 receptor (PLA2R), an antigen expressed in more than 70% of cases of idiopathic membranous nephropathy (IMN), is a biomarker which is now used by physicians for clinical diagnosis. Despite the prevalence of PLA2R in the cases of MN, it is not always effective to use PLA2R for differentiating primary or secondary MNs. On the other hand, urinary albumin assay is one of the de facto tests for kidney function testing for several decades. In this work, urinary albumin species between primary and secondary MN patients are compared using a newly developed capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The distinct patterns of cationic and acidic urinary albumin species, as revealed by this novel CIEF-MS technology, suggest potential applications of this differential analysis for subtyping of membranous nephropathy. Further investigation of these cationic human albumin species in urine may provide clues to the disease onset and development of MN, thus facilitating treatment. In addition, this novel workflow of using CIEF-MS for urinary protein analysis may be beneficial to the research, pathology, prognosis, and diagnosis of many other types of kidney diseases, such as chronic kidney disease, diabetic nephrology, etc. KEY WORDS Membranous Nephropathy, capillary isoelectric focusing – mass spectrometry, Urinary Protein, Albumin
Background Membranous nephropathy （MN）is the leading cause of nephrotic syndrome in adults, affecting ~ 5–10 patients per million population[1]. Although in most patients this disease progresses relatively slowly, about
Journal Pre-proof one-third of the MN patients with nephrotic syndrome eventually develop end-stage renal diseases[2]. With regard to pathology, MN has been considered as one type of immune system complication-related diseases. Primary MN is one type of autoimmune diseases which is caused by in situ binding of circulating antibodies to an endogenous podocytic antigen, leading to the development of subepithelial immune deposits. Since the discovery of M-type phospholipase A2 receptor (PLA2R) as the first major target autoantigen, there is an increasing amount of research being put on the discovery of other antigens for primary MN diseases[3, 4]. A number of recent literatures show that, the levels of circulating anti-PLA2R antibodies play critical roles in the diagnosis and prognosis of primary MN[5]. Deposit these progresses, there are remaining questions that need to be addressed on. First, the mechanism of MN occurring is not thoroughly understood. The role of PLA2R in disease development is an ongoing research area. Besides PLA2R, there are several other novel antigens that have been recently discovered, indicating the underlying complexity of MN diseases, as well as suggesting the possible existence of many subtypes of MN diseases. To this end, Hanna
of
et al. identified cationic bovine serum albumin (cBSA) as one of the MN antigens by immunoassay study. Further studies showed that anti-cBSA antibodies were found in 2% of the MN patients[6]. These patients’
ro
diseases development and prognosis could be identified among groups of patients with other subtypes. It was proposed that, anti-cBSA antibodies bind these cBSA molecules at the anionic glomerular capillary
-p
wall, including the formation of immune complexes. However, there are missing steps in the proposed progression. In particular, there is lack of explanation for the production of anti-cBSA antibodies. Due to
re
limited evidence so far, these newly discovered biomarkers have yet to be used by physicians for diagnosis. In contrast, there are currently at least 30% of the MN patients being left with absence of PLA2R
lP
antibodies[3].
Second, although PLA2R has been an effective biomarker for MN diagnosis, it remains challenging to use it for disease subtyping. Even for primary and secondary MN patients, there is a lack of specificity in the
na
PLA2R testing. As a result, subtyping of MN patients are, at this point, mainly based on clinical experiences. Due to inaccurate and often subjective disease subtyping, the treatment of MN is not straightforward, affecting the effectiveness of the treatment. Meanwhile, recent research of rituximab therapy, which is
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regarded as the most efficient clinical treatment for MN patients, reports independence of baseline proteinuria, pharmacokinetic studies and evaluation of B and T cell subsets[7, 8]. The identification of antiPLA2R antibodies alone is not sufficient to predict the prognosis of the MN patients. Elevated level of albumin in urine samples has been used as one of the widely accepted clinical assays for MN diseases, as well as for many other forms of kidney diseases, for decades[9]. It is well known that, when MN occurs and develops, albumin often leaks into urine and becomes the major protein component in the patient’s urine. Therefore, human serum albumin (HSA) abundance in the urine samples has been widely used as one of the metrics for MNs diagnosis and therapy efficacy monitoring. We report a direct comparison of urinary HSA composition, including the relative abundance of each HSA species, between primary and secondary MN patients, using both reversed-phase liquid chromatography – mass spectrometry (LC-MS) technology and a novel online capillary isoelectric focusing – mass spectrometry (CIEF-MS) technology. The novel CIEF-MS workflow is implemented using an electrokinetically pumped sheath-flow nanospray capillary electrophoresis – mass spectrometry (CE-MS) coupling technology. With this powerful CIEF-MS method, the albumin species in each patient’s urine sample were separated according to their isoelectric point (pI) values, then introduced into high-resolution time-of-flight mass spectrometry for subsequent identification of each species by intact masses. This novel CIEF-MS approach provides two-dimensional information regarding the albumin species in patient’s urine: the pI distribution of the albumin species, as well as the molecular weight of each separated albumin species. In addition, this CIEF-MS technology allows for the separation and identification of minute
Journal Pre-proof urinary albumin variants among patients, whereas the regular LC-MS approach does not offer such separation power. With the successful application of this CIEF-MS workflow for urinary albumin analysis, we report the separation, identification and mass assignment of multiple basic and acidic HSA species from secondary MN patient’s urine sample. In contrast, the primary MN patient’s urine sample does not contain these albumin charge variant species. This study is a proof-of-concept demonstration of the potential application of the newly developed electrokinetically pumped nanospray CE-MS technology for direct human urine analysis. Method Materials. The carrier ampholytes, Pharmalyte® with pH ranges of 3.0−8.0 and 3.0−10.0, were purchased from GE Healthcare Life Sciences (USP, Pittsburgh, PA). LC-MS grade reagents, including water, formic acid, ammonium hydroxide, acetonitrile, and methanol were obtained from Millipore Sigma (Burlington, MA). The electrospray emitters for online cIEF-MS analysis (1.0 mm o.d., 0.75 mm i.d., 15−25 μm tip size) and
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neutral coating PS2 separation capillaries (360 μm o.d., 50 μm i.d.) were supplied by CMP Scientific Corp. The CR3520 CIEF-MS reagent kit is provided by CMP Scientific Corp.
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The urine samples used for this study were collected at Beijing Hospital. The ethical policy of Beijing Hospital was followed for the procedure.
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Urine sample pretreatment. 400 µL of each urine sample was desalted using the Millipore 10K filtering device by 15 min, 14,000 rpm centrifugation, twice at room temperature.
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UPLC-HRMS albumin analysis. A Waters G2-Si Q-TOF HRMS (Waters, Massachusetts, USA) and BEH C4 reversed-phase protein separation was used for the LC-MS study. The flow rate was 40 µL/min.
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Separation was carried out with the following gradient: 15% acetonitrile aqueous solution, hold for 1min; acetonitrile concentration was then risen to 75% within 10 min; after that, the acetonitrile concentration was further increased to 85% in 5 min; the mobile phase was then brought back to initial condition within 4 min.
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The mass spectrometry source temperature was set at 150 ℃. ESI voltage was set at 4.0 kV. Mass range for data acquisition was 300 - 2,000 m/z.
Online capillary isoelectric focusing-mass spectrometry (CIEF-MS). An EMASS-II CE-MS ion source
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(CMP Scientific Corp, Brooklyn, NY, USA) was used to couple the Agilent 7100 capillary electrophoresis with the Agilent 6230 TOF mass spectrometer. Separation capillaries with a length of 75 cm were used throughout the study, in order to balance between analysis time, albumin charge variants resolution, and sensitivity. The Buffer B in the CR3520 CIEF-MS kit (CMP Scientific Corp) was used as the catholyte. The anolyte was Buffer A in the CR3520 CIEF-MS kit. The sheath liquid was the Buffer SL in the CR3520 CIEF-MS kit. Protein samples (0.1−1.0 mg/mL) were prepared in 1.5% Pharmalyte® 3-10 with an equal volume mixing with the Sample Buffer S35 in the CR3520 kit. The catholyte solution was injected under 950 mbar for 20 s, which was followed by sample injection under 950 mbar for 45 s. After catholyte and sample injection, a voltage was applied under normal polarity with a field strength of 250 V/cm. A small pressure of 5-35 mbar was applied at the capillary inlet (anode) to assist mobilization. The electrospray ionization voltage was set at 2.4 kV using the external high voltage power supply that came with the EMASS-II ion source. The distance from the emitter tip to the mass spectrometer was adjusted to 4 mm with the help of a microscope camera. MS Parameters. The capillary voltage of the TOF was set at 0 V. Drying gas was 2 L/min at 350 °C. Fragmentor voltage was 200 V. Acquisition range was m/z 700−4,500. Acquisition rate was 1 spectrum per second. The CE-MS method setup, data acquisition, and analysis were carried out using Agilent MassHunter Workstation software (v B.10).
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Results and discussion Sources of the urine samples. As shown in Fig.1, based on biopsy results, both patients under investigation were PLA2R antibody-positive cases. Their total urine protein concentrations were measured to be 2.119 g/L and 2.570 g/L, respectively. However, the clinical presentation of these two patients were vastly different. The 24h total urinary protein of the Patient A was 1.90 g (urine volume 900 mL). The Patient B had a 24-h total urinary protein of 9.77 g (urine volume 3,800 mL). The urinary samples before buffer exchange have protein concentrations of 2.11 and 2.57 mg/mL, for Patient A and Patient B, respectively. While the Patient A was diagnosed as MN secondary to hypereosinophilia syndrome, this urine protein abundance alone is not uncommon, given that the total urinary protein assay provides limited information about disease subtyping and progression in MN. Based on combined clinical testing results, these two
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patients received different treatments. The Patient A received glucocorticoids therapy alone, and PLA2R antibody transformed negative within a month. The Patient B received tacrolimus therapy and achieved partial remission and significant decrease of PLA2R antibody in the sixth month. Details of these two
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patients are listed in Table 1.
Patient A
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Table 1. Clinical diagnosis information of the two MN patients under investigation Patient B
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Gender
Female
Male
MN subtypes
Secondary MN
Primary MN
PLA2R (RU/mL)
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Urine protein (g/24 h)
90
131
1.9
9.77
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Creatinine (µmol/L)
54
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Age (years)
265.82
167.98
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LC-MS albumin analysis. Urinary proteins from the two patients were analyzed by LC-MS. As shown in Fig.1, albumin was identified as the major protein component in these urine samples. Although the albumin species of both two patients’ urine were separated into two peaks, as shown in the LC-MS data, there was little difference between peak 1 and peak 2, based on their mass spectra. The albumin species in these two urine samples showed differences in their molecular weights. In the urine sample of the Patient A, the albumin species are identified as a single proteoform. However, in the urine sample of the Patient B, there were four distinct albumin species, with deconvoluted masses of 66,436 Da, 66,557 Da, 66,659 Da, and 66,718 Da. The difference in albumin species in the urine samples may be associated with MN subtypes.
Journal Pre-proof Fig.1 Averaged mass spectra of urine albumin species identified by LC-MS. A, mass spectrum of albumin in the urine sample of the Patient A; B, mass spectrum of albumin in the urine sample of the Patient B. The insets are zoom views of the mass spectral peaks, demonstrating the distinct features of albumin species in each patient’s urine sample. CIEF-MS separation condition optimization. In addition to the LC-MS analysis, a recently developed online capillary isoelectric focusing – mass spectrometry technology, CIEF-MS, was applied for further analysis of the urinary albumin species. This online CIEF-MS technology, which is based on a novel electrokinetically pumped sheath-flow nanospray CE-MS coupling technology, is a powerful protein separation and detection system for disclosing protein charge variants[10-13]. In order to facilitate albumin analysis in urine samples, a number of experimental conditions of this CIEF-MS workflow are taken from previous system optimization studies. The effects of some of the critical CIEF-MS parameters, including the duration of catholyte and sample injection, system pressure at the capillary inlet, sample concentration,
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etc., were investigated in previous studies[14]. According to previous findings, the pressure applied at the capillary inlet during the focusing and mobilization process may affect the CIEF-MS resolution and
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sensitivity in many ways. Figure 2 shows the comparison of various pressure levels at the capillary inlet. When 50 mbar was applied, the analysis was completed within 20 min. Whereas, under the 15 mbar
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condition, the entire analysis took more than 50 min. However, fast analysis was not without the cost of compromised performance. For example, while the basic albumin peak was easily distinguished from main
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peak in the injection with 15 mbar running pressure, this basic albumin peak coeluted with the main peak in the analysis when 50 mbar of running pressure was applied. An optimized system pressure of 15 mbar
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was then used throughout this study.
Fig.2 Total ion electropherograms of the CIEF-MS results with various running pressures being applied at the capillary inlet: 15 mbar (Panel A), 25 mbar (Panel B), and 50 mbar (Panel C). Urine sample analysis with CIEF-MS. Proteins isolated from the urine samples were analyzed with the above optimized CIEF-MS conditions. The major protein component was detected to be albumin species in both patients’ urine samples (Fig. 3). There are clear differences on albumin charge heterogeneity patterns between these urine samples. The urinary albumin from the Patient A is present in three charge forms: one basic peak, a main peak, and an acidic peak. As a comparison, only a main albumin peak was detected in the urine sample of the Patient B.
Journal Pre-proof Fig.3 Online CIEF-MS results of the urinary proteins from MN patients. A, results of the urine sample from the Patient A; B, results of the urine sample from the Patient B. Figure 4 shows the averaged mass spectra of each identified albumin species. The three albumin charge variants in the urine sample from the Patient A showed similar m/z mass spectra, with deconvoluted albumin masses around 66,555 Da. The albumin peak in the urine sample from the Patient B (Fig. 4D) showed a deconvoluted mass of 66,560 Da, which matching the LC-MS data quite well. Besides the main species, there are three additional albumin species as identified by unique deconvoluted masses: 66,443 Da, 66,651 Da, and 66,719 Da （Figure SI). These observations may suggest the existence of protein misfolding and post-translational modifications that are associated with the pathology of primary and secondary
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membranous nephropathy.
Fig.4 Averaged mass spectra of the CIEF-MS identified albumin species in the urine samples. Panels A, B, and C:
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averaged mass spectra of the basic, main, and acidic peaks of the albumin charge variants in the urine sample from the Patient A. Panel D: averaged mass spectra of the albumin peak in the urine sample from the Patient B. The insets
Conclusions
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show the deconvoluted masses of each identified albumin species.
Membranous nephropathy is a complex disease and may occur in many forms of subtypes, which come with
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different pathology thus necessitating differential therapy. There is a rise in the demand for membranous nephropathy subtyping at the molecular level. Although anti-PLA2R used to be a sensitive and specific biomarker of primary MN, it may also be tested positive in the cases of secondary membranous nephropathy. As for the patient A reported above, anti-PLA2R rapidly conversed negative after glucocorticoids monotherapy, to which the therapy for primary MN will not react. We made a definite diagnosis for the case of secondary MN. In this case, diagnostic specificity of anti-PLA2R in differentiating primary MN from secondary forms was poor, suggesting an urgent need for new biomarker discovery. We report the analysis of urinary proteins from primary and secondary membranous nephropathy patients, using both regular LC-MS workflow, and a novel online CIEF-MS technology, which was developed by the authors. Compared with LC-MS method, the CIEF-MS approach that we developed showed higher resolution in terms of separating out various urinary albumin species based on their isoelectric points. In this novel CIEFMS-based urinary protein analysis workflow, both molecular weights and protein charge heterogeneity distribution information can be obtained in a fully automated manner. Both cationic and anionic human serum albumin species were identified in the urine sample of a secondary membranous nephropathy patient. This has not been previously reported in the literatures. Besides the uncovering of albumin charge variants in the secondary membranous nephropathy patient’s urine samples, clear difference on the urinary albumin species were discovered between primary and secondary MN patients by both CIEF-MS and LC-MS analysis. The differences in albumin compositions could be associated with diseases pathology. One hypothesis would be
Journal Pre-proof that, the urinary albumin may result from protein misfolding and post-translational modifications, which are modulated by membranous nephropathy disease onset and subsequent progression and development. The accurate subtyping of membranous nephropathy patients remains a challenge, even though there has been a great deal of breakthroughs, such as the discovery of PLA2R antibody. This novel CIEF-MS workflow of urinary albumin charge heterogeneity characterization, as demonstrated by this case study, may lead to a powerful diagnosis tool for distinguishing between primary and secondary membranous nephropathy patients, thus facilitating the decision-making process for physicians. To this end, future investigation which involves large cohort study of primary and secondary membranous nephropathy patients is going to provide valuable information, with regard to the effectiveness of applying this novel CIEF-MS-based urinary albumin charge heterogeneity assay, for the triage of membranous nephropathy patients.
AUTHOR INFORMATION
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Corresponding Author
Fax: +86 10 62331029; E-mail: [email protected]
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**Corresponding author. Tel.: +86 10 85133880;
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*Corresponding author. Tel.: +86 10 62331029;
Fax: +86 10 85133880; E-mail: [email protected]
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Author Contributions ‡These authors contributed equally.
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ACKNOWLEDGMENTS
This work was supported by a grant from Beijing Hospital Nova Project (BJ-2018-131), the National Natural Science Foundation of China (no.81200523), open fund of the state key laboratory of coal resources and safe mining
Foundation (6184049).
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