Body fluid proteome is the most informative proteome from a medical viewpoint. But the lack of accurate quantitation method for complicated body fluid limited its application in disease research and biomarker discovery. To address this problem, we introduced a novel strategy, in which SILAC-labeled mouse serum was used as internal standard for human serum and urine proteome analysis. The SILAC-labeled mouse serum was mixed with human serum and urine, and multidimensional separation coupled with tandem mass spectrometry (IEF-LC-MS/MS) analysis was performed. The shared peptides between two species were quantified by their SILAC pairs, and the human-only peptides were quantified by mouse peptides with coelution. The comparison for the results from two replicate experiments indicated the high repeatability of our strategy. Then the urine from Immunoglobulin A nephropathy patients treated and untreated was compared by this quantitation strategy. Fifty-three peptides were found to be significantly changed between two groups, including both known diagnostic markers for IgAN and novel candidates, such as Complement C3, Albumin, VDBP, ApoA,1 and IGFBP7. In conclusion, we have developed a practical and accurate quantitation strategy for comparison of complicated human body fluid proteome. The results from such strategy could provide potential disease-related biomarkers for evaluation of treatment.
Body fluid proteome generally is the most informative proteome from a medical viewpoint. Almost all tissues in the body communicate with body fluid and release their contents into it, especially upon damage or death [
Multiple analytical approaches have been developed based on separation of proteins in-gel (2DE, DIGE) or gel-free platform utilizing various methods including labeling (ICAT, 18O-labeling, iTRAQ) and label-free strategy. Both of these methods were widely used in quantitative proteomics analysis but also had some limitations. Label-free quantification was quite simple and straightforward, but the precision and accuracy were limited [
Stable-isotope labeling by amino acids in cell culture (SILAC) method provided a comprehensive, robust, and accurate solution for quantitative proteomics analysis. But it is thought to be unsuitable for analyzing tissue and body fluid samples. Hence, a lot of other metabolic labeling methods were introduced in an alternative way such as protein standard absolute quantification (PSAQ), stable isotope-labeled proteome (SILAP), and secretome-derived isotopic tag (SDIT) strategies. However, PSAQ uses full-length isotope-labeled proteins as isotope-dilution standards for MS-based quantification of target proteins in complex matrices. It was difficult for the accurate quantitation of large numbers of serum proteins. In addition, it seems more appropriate for the combination of PSAQ and SRM (PSAQ-SRM) to allow highly accurate biomarker quantitation in serum samples. Serum mixed with SILAC-labeled secretome of culture cells (SILAP and SDIT) was easy to manipulate and quantify large numbers of serum proteins. However, serum contains all tissue proteomes as subsets. It was not adequate to use one or several kinds SILAC labeled secretome of cultured cells as standards in quantitative serum proteomic studies.
In this paper, we have proved that the serum from SILAC mouse could also be used as internal standards to quantify human serum and urine. With this novel strategy, we performed the quantitative proteomics analysis by extensive multidimensional separation coupled with tandem mass spectrometry (IEF-LC-MS/MS) for the urine from Immunoglobulin A nephropathy (IgAN) patients treated and untreated. As the quantification standards display the same biochemical features as urinary samples, they can be spiked into the samples at early stages of the analytical process, which can avoid differences that yielded in digestion and nonspecific losses suffered during extensive sample processing between internal standard SILAC mouse and human samples. In conclusion, our research provided a comprehensive and accurate solution for human body fluid analysis.
Urea, tris, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), ammonium bicarbonate (NH4HCO3), and iodoacetamide (IAA) were purchased from Bio-Bad (Hercules, CA). The Lys-C enzyme was purchased from Wako (Osaka, Japan). Acetonitrile (HPLC grade) was obtained from Merck (Darmstadt, Germany). Formic acid was obtained from Aldrich (Milwaukee, WI, USA). All the water used in the experiments was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). The 13C6-lysine labeled mouse serum (MT-LYSC6-MSE) was purchased from Cambridge Isotope Laboratories, Inc.
Immediately after collection, fasting blood samples of healthy subjects were allowed to clot at room temperature for four hours, and the serum was collected and centrifugated at 3000 rpm/min for 15 min. Informed consent was obtained from each person in written format and approved by Longhua Hospital, Shanghai University of Traditional Chinese Medicine Review Committee.
The 24 h urine samples were collected from six healthy subjects. Informed consent was obtained from each person in written format and approved by Longhua Hospital, Shanghai University of Traditional Chinese Medicine Review Committee. Volume of 200 ml urine from each person was concentrated by the acetone precipitation approach [
The urine from IgAN patients with and without glucocorticosteroid treatment was collected and prepared as mentioned earlier for urine from healthy persons [
The sample digestion was processed by FASP procedure as described previously [
The p
A Surveyor liquid chromatography system (Thermo Finnigan, San Jose, CA, USA), consisting of degasser, MS Pump, and auto sampler, equipped with an analytical C18 column (RP, 75 µm × 150 mm, Column Technology Inc., CA, USA) was used. The HPLC solvents used were 0.1% formic acid (v/v) aqueous (A) and 0.1% formic acid (v/v) acetonitrile (B). The reversed-phase gradient was from 2% to 40% mobile phase B in 180 min at 120 µL/min flow rate before the split and 250 nL/min after the split. A linear ion trap/Orbitrap (LTQ-Orbitrap) hybrid mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an NSI nanospray source was used to for MS/MS experiment with ion transfer capillary of 200°C and NSI voltage of 1.80 kv. Normalized collision energy was 35.0. The mass spectrometer was set that one full MS scan (m/z 400–1800) was acquired in the Orbitrap parallel ten MS/MS scans in the linear ion trap on the ten most intense ions from the full MS spectrum with the following Dynamic Exclusion settings: repeat count 2, repeat duration 30 seconds, and exclusion duration 90 seconds. The resolving power of the Orbitrap mass analyzer was set at 60,000 for the precursor ion scans (m/Δm 50% at m/z 400). The m/z (445.120025) was used as an internal lock mass and calibrant ions in the full MS scan.
All raw mass spectrometric data were analyzed with the MaxQuant software (version 1.3.0.5 [
In quantification, Lys6 in MaxQuant software was selected as heavy internal standard to get the intensity for heavy and light peptide and the SILAC ratio. The peptides shared between human and mouse were quantified by their SILAC pairs, and the human-only peptides were quantified by mouse peptides with nearest retention time.
All statistical analyses were conducted in the R environment (
SILAC method provided a comprehensive, robust, and accurate solution for quantitative proteomics analysis. And the development of SILAC mouse expanded its application into tissue and body fluid samples [
The major obstacle for the application of SILAC mouse in human samples was the different protein sequence between the two species. But as a necessary step of protein digestion in bottom-up proteomics, the compared results were in fact not protein sequences but digested peptide sequences, which increased the possibility for human sequence to find corresponding SILAC pair in labeled mouse internal standard. Even the proteins with different sequence between human and mouse may have consistent digested peptides and could be quantified. Hence, we proposed a novel workflow, in which the SILAC-labeled mouse serum was used as internal standard to quantify human body fluid (Figure
The experiment design and theoretic digestion result. (a) The serum of SILAC mouse was mixed with serum and urine from human, and then enzyme digestion was performed. The shared digested peptides between human and mouse could be selected as SILAC pair and quantified. (b) The comparison of molecular weight distribution for the theoretic digestion peptides of trypsin and Lys-C. (c) The comparison of Lys-C theoretic digestion peptides in human and mouse database.
As the serum of SILAC mouse was labeled by lysine, the lysine-containing peptides from human samples and mouse internal standard could be distinguished in MS analysis, and the peptides without lysine were undistinguishable and helpless for quantitative analysis. To find the enzyme with better performance for such kind of experiment design, trypsin and Lys-C theoretic digestions for human IPI proteome database were performed and compared. As a result, Lys-C digestion produced more lysine-containing peptides in almost every molecular weight section (Figure
Based on the results from theoretic digestions, there were still many human-only peptides. To make better use of these peptides, we divided the quantified peptides into three parts (Figure
The demonstration for peptide quantification. The peptides were divided into 3 groups. The shared peptides between two species were quantified by traditional SILAC. The mouse-only peptides were taken as reference. And the human-only peptides were compared with co-elution reference peptides to get the quantification results.
To ensure the feasibility of our quantitation strategy, preliminary experiment with human serum and urine samples were performed. Initially, the serum and urine from healthy human subjects were collected and mixed with isotopic-labeled mouse serum, respectively. And two technical replications for each sample were performed. Then, peptide quantification was done as illustrated in Figure
The quantitation results in preliminary experiment. (a) The ratio distribution of shared peptides. (b) The scatter plot and correlation coefficient for quantitation results of shared peptides. (c) The ratio distribution of shared peptides in comparison of replication experiments. (d) The ratio distribution of human-only peptides. (e) The scatter plot and correlation coefficient for quantitation results of human-only peptides. (f) The ratio distribution of human-only peptides in comparison of replication experiments.
For the shared peptides, the ratios distributions (Figure
The analysis for human-only peptides also showed advantageous result. In ratios distributions, there was a little shift for serum ratios and no obvious peak for urine ratios (Figure
IgA nephropathy is the most frequent type of glomerulonephritis and characterized at biopsy by a wide variability of features. Baseline proteinuria is one of the main predictors of IgAN progression. But few studies have evaluated whether some other components of proteinuria could improve the prediction of IgAN and evaluate the effect of therapeutic approaches.
Here the novel strategy was applied in the urine samples from IgAN patients treated and untreated. There were totally 3 patients, and two technical replications for each sample were performed (Figure
The experiment design for comparison of IgA patients’ urine untreated and treated. The UT in the end of sample name indicated that the sample was untreated, and T in the end of sample name indicated that the sample was treated.
The quantification results were also from shared peptides and human-only peptides, and the mouse-only peptides found in all samples were used as quantification reference (Figure
The quantitation results in analysis of IgA patients’ urine. (a) The ratio distribution of shared peptides in comparison with replication experiments. (b) The hierarchical cluster analysis result of shared peptides. (c) The principal component analysis result of shared peptides. (d) The ratio distribution of human-only peptides in comparison with replication experiments. (e) The hierarchical cluster analysis result of human-only peptides. (f) The principal component analysis result of human-only peptides.
Then, the limma package in R environment was used to find significant changed peptides between two groups. As a result, 9 and 44 peptides were selected, respectively, in shared peptides and human-only peptides with
To further analyze the proteins influenced by the treatment and reveal their association with IgA nephropathy, a detailed literature search was performed. And the significant proteins associated with nephropathy were listed in Table
The significant proteins associated with nephropathy.
Peptide sequence | Protein name | Fold changes treated : untreated | Relevance with nephropathy | Reference |
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ACEPGVDYVYK | Complement C3 | −3.3 | Reported as a potentially novel predictor of progressive IgA nephropathy | [ |
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HEVTGWVLVSPLSK | Insulin-like growth factor-binding protein 7 | −3.2 | The urinary levels of other IGFBPs correlated with the development of renal disease | [ |
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VHTECCHGDLLECADDRADLAK | Albumin | −2.3 | Most famous indicator for nephropathy | [ |
YICENQDSISSKLK | −2.0 | |||
AEFAEVSK | −1.9 | |||
AAFTECCQAADKAACLLPK | −1.6 | |||
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HQPQEFPTYVEPTNDEICEAFRK | Vitamin D-binding protein isoform 3 | −2.1 | Enhanced excretion in urine during diabetic nephropathy | [ |
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DSGRDYVSQFEGSALGK | Apolipoprotein A-I | −2.1 | Increased in the plasma of diabetic nephropathy patients | [ |
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HYYIGIIETTWDYASDHGEK | Ceruloplasmin | −1.7 | Enhanced excretion in urine during diabetic nephropathy | [ |
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SSFVAPLEK | Pigment epithelium-derived factor | −1.6 | A urinary marker for diabetic nephropathy | [ |
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DSAHGFLK | Transferrin | −1.6 | Enhanced excretion in urine during diabetic nephropathy | [ |
Complement C3 is the most significant decreased protein after treatment in our result. IgA nephropathy is characterized by IgA deposition, and it is frequently accompanied by complement C3. The serum IgA/C3 ratio is taken as a diagnostic marker for the progression of IgAN [
Albumin is the most famous indicator for renal disease [
Besides, we have also found four proteins, which are vitamin D binding protein [
On the other side, there were some proteins of overexcretion in the urine of treatment group, including Annexin A1, Cystatin-A, Angiotensinogen, Gamma actin-like protein, and Zinc-alpha-2-glycoprotein. Most of these proteins had never been reported as nephropathy biomarkers or only found to be significant in large-scale expression analysis [
In this work, we proved that SILAC-labeled mouse serum could be used as internal standard for human body fluid proteome analysis. It provided comprehensive and reliable quantitative results and could specifically address the obstacles in this field. With the application of this strategy in patient samples, we can obtain valuable findings in biomarkers discovery for monitoring disease progression and evaluating therapeutic efficacies.
S. Zhao, R. Li, and X. Cai contributed equally to this work.
The study is supported by the Foundation (no. SHDC12010114) of Joint Research Project of Shanghai Municipal Level Hospital and Technology Special Projects Major New Drug Development (2011ZX09302-006-04) as well as the Shanghai Government Foundation (10DZ1951202). The Authors gratefully acknowledge the support of SA-SIBS Scholarship Program.