JPROT-01997; No of Pages 11 JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
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Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation
Mathieu Riffault, David Moulin, Laurent Grossin, Didier Mainard, Jacques Magdalou, Jean-Baptiste Vincourt⁎,1
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Ingénierie Moléculaire et Physiopathologie Articulaire, UMR 7365 CNRS-Université de Lorraine, Faculté de Médecine, 9, Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy, France
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Article history:
Proteomics users enjoy the rapid development of LC-MS-based label-free relative
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Received 31 July 2014
quantification methods but in practice these remain restricted to mass spectrometers
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Accepted 22 October 2014
using electrospray ionization. Here, tools dedicated to ion chromatogram extraction, time
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alignment, signal normalization and statistical analysis were used to interpret label-free
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relative difference between primary human chondrocyte secretomes and dilutions thereof, analyzed successively by LC-MALDI. The analysis of secretomes diluted into
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Collagen
culture medium demonstrated that abundant proteins could be relatively quantified
Extracellular matrix
within 1.5–20-fold changes with satisfactory statistics. In addition, comparison of
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Cartilage
multiple samples requires analyzing most samples in TOF mode only, saving considerable
Differentiation
machine-time usage. The method allowed identification and quantification of most
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Phenotype
secreted proteins relevant to the chondrocyte phenotype and evidenced their up- or down
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ProfileAnalysis
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regulations by TGFβ1 and patient-to-patient differential expression. Novel targets of TGFβ1
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were evidenced, such as pro-collagen C-proteinase enhancer protein 1, Metalloproteinase inhibitor 1, Fibulin-3, Tetranectin and Cartilage Intermediate Layer Protein 1, while others
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Secretome TGFβ1
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Keywords:
match previous findings. Several were verified by Western blot. This whole workflow is non-invasive, compatible with many cell culture protocols, technically straightforward and rapid, particularly regarding mass spectrometer time usage and could make label-free LC-MALDI analysis of low-complexity proteomes a major tool for routine cell culture characterization. Biological significance The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome
⁎ Corresponding author at: Team 2, Molecular, Cellular, Therapeutic Engineering and Glycosyltransferases, UMR 7365 CNRS-UL, Biopôle, Faculté de Médecine, 9, Avenue de la Forêt de Haye, BP 184, 54505 Vandœuvre-lès-Nancy, France. Tel.: +33 3 83 68 5412; fax: +33 3 83 68 5409. E-mail address:
[email protected] (J.-B. Vincourt). 1 Present address:: Proteomics FR3209, Biopôle, Faculté de Médecine, 9, Avenue de la Forêt de Haye, CS 50184, 54505 Vandœuvre-lès-Nancy, France.
http://dx.doi.org/10.1016/j.jprot.2014.10.026 1874-3919/© 2014 Published by Elsevier B.V.
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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regulation by TGFβ1 and is proposed as a routine tool to assess the quality of biomaterials
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designed for cartilage repair and to quantitatively investigate the influence of environ-
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mental factors upon it.
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Human articular cartilage was procured under general anesthesia during total knee replacements in the context of late osteoarthritic disease (Kellgren and Lawrence grade III to IV). This study was approved by our local Research Institution review board (registration number UF 9757 – CPRC 2004 – Cellules souches et chondrogénèse). Three patients were used in this study and gave written informed consent in accordance with the usual ethical regulations in collaboration with our local bone bank. Two were males and ages were from 53 to 71 at procurement.
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2.1. Tissue procurement
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2. Experimental procedures
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chromatogram fold-changes [22], which then were assigned to the corresponding identified peptides and used to calculate the protein fold-changes. The method was found accurate to directly quantify changes in protein secretion by primary human chondrocytes and used to monitor chondrocyte secretion changes between donors and its regulation by TGFβ1.
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The secreted proteome is relatively simple in its composition and exhibits many specialized functions of individual cell types, including details of intercellular communication and extracellular matrix (re)modeling. In addition, in the context of in vitro cell culture, the soluble secretome is discarded when changing the medium and therefore, its collection is absolutely non-invasive in the vast majority of cultivation systems. This makes the secretome a potentially ideal material to routinely characterize cell cultures in the contexts of biological investigations and quality control of cell cultures of any types. Increasing efforts are being deployed towards qualitative and quantitative characterization of secretomes. Most cells require serum for expansion. However, whenever the response of a cell type to a given soluble mediator is investigated, the stimulation is in most cases performed under serum-free conditions, because serum components interfere with most stimuli. Also, in the expanding field of stem cell-based tissue repair, most protocols use serum-containing media for stem cell growth and pre-orientation, but serum-free media supplemented with minimal exogenous proteins (Insulin, Transferrin) for differentiation into mature cell types [1–3], because it favors differentiation and resolves issues of serum compatibility with in vivo transplantation. The same media are now commonly used to promote, for instance, the differentiated articular chondrocyte phenotype [4]. Under such circumstances, the conditioned medium contains largely enriched secretome, facilitating downstream analysis without particular adaptations. So far, proposed approaches for the quantitative analysis of serum-free secretomes based on mass spectrometry have required either metabolic labeling [5–8] or, to a lesser extent, isobaric labeling [9]. These methods have been efficient; however, metabolic labeling, as click chemistry, remains particularly expensive, requires adaptations of cultivation systems, whereas secretome characterization for quality control could only be achieved under sufficiently robust, straightforward and yet affordable sample preparation and mass spectrometry methods. Since its first description over a decade ago [10,11], MS-based label-free relative quantitation approaches, consisting in measuring ratios of LC-MS peak areas for each individual identified peptide [12,13] has constantly gained in feasibility and, as a consequence, in popularity, due to the established reproducibility of LC systems, the development of adapted mass spectrometry instruments and data interpretation tools [14,15]. In the vast majority of cases, label-free quantification remains restricted to electrospray ionization (ESI) mass spectrometers [16,17]. Yet, several independent laboratories have reported label-free quantitative measurements by MALDI [16,18,19] or LC-MALDI [20]; reviewed in [21]. Here, we have processed LC-MALDI data obtained from consecutive LC-MALDI runs through the most commonly used principles of label-free relative quantification, that is, the calculation of individual ion
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1. Introduction
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© 2014 Published by Elsevier B.V.
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2.2. Cell culture, stimulation and counting
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Articular chondrocytes were prepared strictly as described in [23] in 75 cm2 culture flasks and grown to sub-confluence in DMEM/F12 medium (Invitrogen) containing 15% foetal calf serum. Cell cultures were rinsed twice in PBS and stimulation with 10 ng/ml recombinant human TGFβ1 (R&Dsystems) was performed over 96 hours in serum-free medium supplemented with insulin, transferrin and selenium (ITS, Sigma), a minimal supplement widely used for in vitro stem cell differentiation [1,2], used here to favour the chondrocyte phenotype [4]. Secretomes were collected after 48 hours, medium was replaced and secretomes were collected again after another 48 hours. In order to estimate the effect of TGFβ1 over cell proliferation, cultures collected by trypsin digestion and cells were counted using a Mallassez cell.
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2.3. Soluble secretome processing for in-solution digestion and 146 LC-MALDI 147 From each 75 cm2 flask conditioned medium, a 1 ml aliquot was kept for in-solution digestion. Deoxycholate (DOC) was added at 0.02% for 15 min on ice and trichloroacetic acid (TCA) was added at 15% for 1 hours. One additional sample made of the same volume of pure culture medium was processed. Proteins were pelleted by centrifugation at 16,000 g for 15 min at 4 °C and rinsed twice with ice-cold acetone. Pellets were dried gently and resuspended in 30 μl 6 M urea, 50 mM Tris-HCl (pH 8.0) under sonication (for total protein content measurement, based on absorbance at 280 nm, measured on a Nanodrop spectrophotometer). A fraction corresponding to
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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2.5. Peptide and protein identification
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The pure culture medium was used as a reference to generate an exclusion list which was used afterward for all other samples. One sample of each experimental group was used for identification, being analysed in TOF/TOF mode using a WARP-LC method selecting masses with S/N > 6 in LIFT mode as follows: all RT/mass coordinates of one sample (excluding those contained in the exclusion list) were processed for fragmentation. Then, the RT/mass coordinates found in the first sample were excluded as well from the total compound list of a second sample, which was then processed for fragmentation, and so on until the whole sample list was processed. Then, all identification runs were merged in WARP-LC prior to database search. Peptide assignments
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2.6. Integration of individual peptide fold changes per protein 230 for protein quantification 231
2.4. Individual ion quantitative measurements through LC-MALDI-TOF analysis LC-MALDI runs dedicated to quantification were processed using dedicated automatic methods piloted by WARP-LC software on an Autoflex speed MALDI-TOF/TOF mass spectrometer (Bruker) in the 800-3500 mass range, using nextneighbour external calibration for all MALDI spots. LC runs to be compared to each other were all analysed sequentially for TOF measurements only, using 2000 random laser shots per spot at a 1000 Hz frequency. MALDI-TOF runs were converted to BAF files and imported to ProfileAnalysis 2.0 for time alignment, bucket fold changes and t-test calculation using the following set-up: WARP-LC, time alignment, advanced bucketing using time alignment parameters, quantile normalization, which consists in making distributions of populations similar, a value count of bucket filter > 3, a value count of group attribute within bucket filter > 3 and empty bucket attributes being allowed. These filters allow to take into account only (retention time, RT/mass) coordinates observed in all replicates of a given experimental group for protein relative quantification but also evidence peptides found in one group but absent in another in the Proteinscape 2.1 (Bruker) table (see below).
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were performed from TOF/TOF spectra by Mascot interrogation (Matrix Science) of the Swissprot database piloted and compiled by Proteinscape with a mass tolerance of 50 ppm in TOF mode and 0.8 Da in TOF/TOF mode, with optional cysteine carbamidomethylation, methionine oxidation and allowing one trypsin miss-cleavage. In addition, proline oxidation was allowed in order to maximize peptide identification of collagen species [24] without affecting peptide identifications for other proteins. Only peptides with Mascot scores above 15 were taken into account. Protein identifications were allowed from a minimum of 2 TOF/TOF validated peptides using the decoy peptide library method for FDR calculation, requiring FDR < 1%. However, these criteria were found much less stringent than those below for quantification and therefore were never limiting.
ProfileAnalysis calculations were exported to Proteinscape 2.1 server and linked to protein and peptide lists from identification runs using the label-free quantitation module of proteinscape using 0.3 min retention time and 0.1 Da mass tolerances. Only peptides of unambiguous identity matching statistical quality control criteria defined at the individual ion relative quantification level (above) were taken into account for protein ratio calculation. For each protein quantified by at least four distinct peptides, the two most extreme peptide fold-changes observed were excluded, remaining individual peptide fold changes were used to calculate the median value and their variation was expressed as the relative coefficient of variation (CV) of peptide fold-changes per protein.
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2.7. Experimental set-up
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The present method was developed in the intention of investigating the secretory responses of primary human chondrocytes to TGFβ1. Therefore, its accuracy was assayed using standards which fit the nature of samples to be analyzed afterwards. Chondrocytes obtained from three patients were cultured and stimulated separately as one would have done using other analytical methods and conditioned media were collected after 48 and 96 hours (medium being replaced in between). Stimulations and control cultures were performed in ITS-supplemented medium, which is widely used for mesenchymal stem cells cultivation [1–3] and cartilage biology [4] and contains 15.5 μg/ml proteins (insulin and transferrin) according to manufacturers. Therefore, not only is its composition well suited for chondrogenic differentiation, but its total protein content is almost 3 logs lower than media supplemented with 10% FCS, leaving much more room for secreted protein analysis. Because TGFβ1 is known to promote chondrocyte growth depending on cultivation conditions [25], cell growth was estimated by cell counting after trypsin digestion. All chondrocyte cultures responded to TGFβ1 by growing very moderately (Supplemental data 1). The total protein content of fresh versus 48 hour conditioned media was estimated by spectrophotometry (Supplemental data 1), demonstrating that the content of control chondrocyte culture media was 30-40% above that of fresh medium, while the content of TGFβ1-stimulated
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a maximum of 10 μg total proteins was processed for digestion as follows: Cysteine residues were reduced by addition of 4 mM DTT for 45 min, alkylated by addition of IAA at 40 mM for 30 min and remaining IAA was blocked by addition of 40 mM DTT for 15 min. Protein solutions were diluted 10-fold in 50 mM Tris, pH 8.0, 1 mM CaCl2 and 1 ng trypsin was added per 100 ng protein mixture and digestion was allowed overnight at 37 °C and stopped by adding acetonitrile (ACN) to 2% and trifluoroacetic acid (TFA) to 0.5%. A maximum of 500 ng protein digests were separated by nanoHPLC on an Ultimate3000 system equipped with a 20 μl sample loop, a pepMap 100 C18 desalting precolumn and a 15 cm pepMap RSLC C18 fractionation column (all from Dionex). Samples (5 μl) were injected using the μlpickup mode and eluted by a 2 to 45% ACN gradient over 30 min at 300 nl/min. Fractions (170, 6 seconds each) were collected on a ProteineerFcII (Bruker) over 21 min and elutions were directly mixed on MTP-1536 TF target (Bruker) spots to α-cyano-4-hydroxycinnamic acid (Bruker).
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Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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PRECIPITATION & DIGESTION LC-MALDI-TOF acquision of raw quantave data within 10 min per sample
(oponal) MS/MS ID of compounds if unknown CV between pepdes
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to build a library of samples containing predictable intersample relative fold-changes of all secreted proteins (Fig. 1). Then, the mixtures were processed for digestion and successive, automatized triplicate LC-MALDI-TOF analysis for raw quantitative data acquisition. The pure secretome was analyzed twice in triplicates in order to allow comparison of this sample to itself whilst introducing the same analytical biases as those for inter-sample comparisons. Then, one sample only, corresponding to undiluted conditioned medium was further analyzed through MALDI-TOF-TOF of all (about 1,000) compounds observed in this sample but not in pure fresh medium. Obtained TOF-TOF data were used for peptide/ protein identification as would be done in a regular LC-MALDI analysis. In the meanwhile, LC-MALDI-TOF data were aligned and analyzed through ProfileAnalysis software to calculate
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chondrocytes was 80-150% above. Noticeably, stimulation over 96 hours farther increased total secretion for all three patients. Therefore, the complexity of secretomes collected in ITS-supplemented media falls within an ideal compromise for proteome analysis using LC-MS workflows: on the one hand, secreted proteins constitute a non-negligible fraction of the total mixture, facilitating their detection, identification and quantification, but on the other hand, all collected samples share an important proportion of their components arising from the additives, allowing reproducible separation and, if required, providing landmarks for post-acquisition retention time/mass alignments for proper data interpretation. In the pilot study, conditioned media from primary human chondrocytes stimulated with TGFβ1 for 96 hours were diluted at several ratios into fresh culture medium in order
m/z
m/z
individual mass/RT fold-change with t-test
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Fig. 1 – Summarized workflows of the pilot and biological experiments. Detailed methods for each step are explained in materials and methods. Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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when measuring k-fold changes and was obtained by averaging the theoretical probabilities to do so over the CV population obtained for k-fold expected changes (Supplemental data 2).
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t-tests and sample-to-reference signal fold-changes for all mass/retention time coordinates, the reference being the undiluted secretome. Fold-changes, together with the corresponding t-tests, were assigned to peptides identified from TOF-TOF data (Fig. 1). Therefore, the principle for relative quantification used here are very similar to that more widely utilized for LC-MS data with slight specificities.
3. Results
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2.8. Western blots
3.1. Label-free differential LC-MALDI provides accurate secreted 363 protein fold-change measurements in the 1.5–20-fold range 364
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Separate aliquots of culture medium were precipitated as for LC-MALDI analysis but resuspended directly in Laemli sample buffer containing DTT, boiled and separated through SDS-PAGE on 4–20% gradient acrylamide Criterion gels (Biorad). Transferred PVDF membranes were blocked with BSA and primary antibodies were as follows: monoclonal mouse anti-FN, from Sigma; monoclonal mouse anti-PGS2, from R&D systems; polyclonal goat anti-TF, from Santa Cruz; secondary antibodies were from Cell signaling. Polyclonal chick anti-CPI (C-propeptide of type Iα1 collagen) and monoclonal mouse anti-CPII (C-propeptide of type II collagen) were described previously [26,27]. HRP luminescence was acquired on a GeneGnome camera equipped with Genesnap acquisition software.
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All experiments were performed and analyzed in triplicates. For TGFβ stimulation calculation, LC-MALDI runs were grouped per patient and per experimental condition and compared per patient between experimental conditions. To determine patient-to-patient variations, one group was constituted of the three replicates obtained for a given patient (n = 3) in a given experimental condition and another group was constituted of all three replicates of all three patients in the same experimental condition (n = 9). The FDR is usually estimated based on the average CV, as calculated from the total population of measurements. However, the FDR is calculated for a given ratio threshold and therefore, if the CV value depends on the measured ratio, the global average CV is not representative of the CV at this ratio. For instance, the error measurements observed for 0.1-fold changes (which are at the highest) would contribute to the calculation of the FDR at 0.67-fold changes (which exhibit much lower error rates, as depicted in Fig. 2C and D) and therefore result in their over-estimation. Therefore, both FDR and Lack of Discovery Rates (LDR) were calculated to recapitulate the utility and limits of the method for quantification. Both were calculated using the relationships between CV and the probability of making false measurements established in [28] assuming measurements as normally distributed variables. The FDR was considered as the rate of false k-fold change observations when measuring 1-fold changes at a given k threshold. The theoretical probability that two replicate measurements differ by a factor k or more [p(k), as termed in [28]] was calculated for each CV value available at ratio = 1 and the FDR at a k threshold was considered as the average of all obtained p(k) (Supplemental data 2). Conversely, the LDR at a k threshold was considered as the rate of false 1-fold change observations
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3.2. Precision and deviation from the expected ratio are 376 affected much more by the measured ratio than the number 377 of available peptides 378
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In the pilot study, protein identification provided a list of 35 proteins using an FDR of 1%. Among them, however, not all were assigned relative quantifications because none of their peptides passed the quality control criteria (as assigned in ProfileAnalysis and described in Methods). The vast majority of the measurements fitted closely to expectation (Supplemental data 2). All quantification data obtained for each protein at each dilution rate were averaged per dilution rate and plotted against the corresponding dilution rate (Fig. 2A). Observed ratios closely matched expectations with relatively good correlation.
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When it comes to protein relative quantification, the debate of how many and which peptides of the protein should be used to assess protein ratio remains open [22]. It is generally considered, however, that a minimum of three reference peptides is highly preferable. Here, however, the relative CVs which were calculated for all protein measurements from all available peptides in the pilot experiments were not found to depend primarily on the number of peptides used (Fig. 2B); nor did the relative deviations from expectations (Supplemental data 2). Therefore, given that even a protein measurement made from a single peptide ratio actually corresponds to a triplicate measurement in both groups to be compared (as set-up in the quantitative ion quality control filters, which allow a peptide to be used for quantification only if it has been observed in all three replicates of each group) and given that the statistics over this peptide ratio are provided in the protein table, it make sense to allow protein measurement even from a single peptide as long as the quantitative statistics of its ratio and the confidence in its identification (and that of its parent protein) are acceptable. On the other hand, both relative CVs and deviations dramatically spread as expected protein ratios decreased (Fig. 2C and D), indicating that both the precision and accuracy of the measurement depended on the ratio itself. As explained in methods, this led us to consider not only the FDR, but also the Lack of Discovery Rate (LDR) as reflections of the robustness of the method. Indeed, because the CVs are extremely low when measuring a fold-change close to 1, the FDR comes down to 0.3% if using a threshold of 1.5-fold (Supplemental data 2), indicating that falsely measuring a 1.5-fold change for a protein which actually is not regulated is extremely unlikely. On the other hand, the LDR at the same 1.5-fold threshold rises up to 10%, indicating that missing a 1.5-fold change may theoretically
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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occur for 1 out of 10 proteins. Thus, the proportion of false positive measurements is extremely low using the present method, but that of false negatives (which is very rarely taken into account in other studies) is only fair.
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Fig. 2 – Label-free differential LC MALDI allows relative quantification of soluble secreted proteins. (A) Average ratio measurements for all measurable proteins plotted against the theoretical ratios in each mixture of the pilot experiment. Error bars: global SD between proteins. The linear regression was calculated assuming proportionality. r2: determination coefficient of the regression. Numbers above the line indicate the number of protein measurements used at each ratio. (B, C) Distribution of the coefficients of variation (CV) of measurements obtained for all proteins at all dilution rates as functions of (B) the peptide number available for calculation and (C) the expected ratio. (D) Distribution of the CV of measurements obtained for all proteins at all dilution rates as functions of the expected ratio. Black squares: individual protein relative error; white squares: average error over all protein measured at the indicated ratio. Comparison to (C) suggests that the relative error and CV are actually intimately correlated and both function of the ratio.
The developed method was then used to investigate changes in the secretome of primary human chondrocytes obtained from 3 separate donors upon TGFβ1 stimulation for 48–96 hours. As explained above, chondrocyte growth was minimally affected by TGFβ1 stimulation. Therefore, equivalent volume fractions were precipitated and processed for in-solution digestion and direct LC-MALDI analysis. Data were analyzed in three distinct comparisons: chondrocytes of each patient stimulated for 48 or 96 hours were compared to un-stimulated chondrocytes from the same patient at the same time-point (Fig. 3) and patients were compared to each other without stimulation (Supplemental data 3&6) or after stimulation for 48–96 hours
(Supplemental data 4 and 7). The CV repartitions were much more advantageous for patient-to-patient comparisons than for stimulation effect measurements, in agreement with the above-described dependence of CV on measured ratios (Fig. 1). Upon TGFβ1 stimulation for 48 hours, the vast majority of regulations were found conserved between patients. 15 out of 30 proteins were found modulated more than 1.5-fold in all three patients (Supplemental data 5). Two proteins (CILP1 and PRG4) were found induced for all three donors but their regulation could not be quantified because their levels were not measurable in control samples. Down-regulations were all found moderate, never exceeding 2-fold in all patients. Up-regulations observed after 48 hours of stimulation were all confirmed and most of them were farther increased after 96 hours (Fig. 3). In contrast, proteins found down-regulated after 48 hours still exhibited moderate regulations after 96 hours. Five additional proteins could be detected after 96 hours in at least one experimental group. However, the rate of proteins for which a regulation could be evidenced but not measured rose to roughly one third, indicating profound
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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REGULATION AT 48H PATIENT 1
PATIENT 2
REGULATION AT 96H
PATIENT 3
Acc. N° fold reg CV fold reg CV fold reg CV
PATIENT 1
GLOBAL AVE
CV AVE PEPT #
PCOC1
CLUS
PGS2
CO6A1
LUM
PGCA
CO6A1
PGS2
CO2A1
MGP
CH3L1
COBA1
CLUS
HPLN1
TIMP1
CH3L1
PA2GA
COCA1
FSTL1
CO2A1
MGP
CO1A2
FMOD
FMOD
SPRC
CO3A1
BGH3
TIMP1
CO1A2
CO1A1
CO3A1
SPRC
[32]
CO1A1
PA2GA
[41] [35] [33] [30]
TENA
BGH3 PGS1 COMP
COMP PGS1 HTRA1
IBP3
FINC
LTBP2
IBP3
TENA
[29] [36]
PRG4
[37]
TETN
AEBP1
CILP1
IBP7
PRG4
[31]
[39]
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FINC
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CFAH
CV AVE PEPT #
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FBLN3
R O
PGCA
FBLN3
AVE
[34]
HEMO
PCOC1
GLOBAL
PATIENT 3
Acc. N° fold reg CV fold reg CV fold reg CV
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LUM
PATIENT 2
RELATIVE CV > 20%
1
1
none
< 10%
>2
>1.5 no data available
TSP1
CTGF
[40]
E
up
[38]
LTBP2
C
down
CO5A1
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REGULATION FOLD
E
CILP1 AVERAGE PEPTIDE # (over all 3 donors)
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regulation by TGFβ1. The secretomes obtained after 96 hours were analyzed by western blot for the proteins against which we had available antibodies (Fig. 4) and these were in a general good agreement with our mass spectrometry data.
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Fig. 3 – Regulation of secretion levels by TGFβ1. Modulation of secretion levels of individual proteins upon TGFβ1 stimulation for 48 or 96 hours. Proteins were annotated with their Swissprot abbreviated name and listed per increasing measured fold change. Down: indicates that peptides corresponding to the protein were detected only in control secretomes; none: the measured ratio was found comprised between 0.67 and 1.5 and was therefore considered as insignificant; up: peptides corresponding to the protein were detected only in the secretome of stimulated chondrocytes. Global regulation was considered as the average of regulations observed from the 3 donors.
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4. Discussion
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TGFβ1, the most studied member of the TGFβ family, exerts strong but context-dependent regulation over extra-cellular matrix synthesis. It strongly induces the extracellular matrix synthesis by chondrocytes and is widely used as supplement in serum-free, minimally supplemented culture media to promote stem cell differentiation into chondrocytes or osteoblasts [1,3]. Although its functions act at either transcriptional or post-transcriptional levels [32], its targets have never been investigated directly through proteomics approaches. In order to demonstrate the efficacy of MALDI-based, label-free relative
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quantification, we used the approach to compare secretomes from control versus TGFβ1-stimulated human primary chondrocytes grown in the same medium as that classically used for stem cell differentiation. Many of the proteins found regulated here at the proteomic level had been identified by other means previously in similar or different cellular models (annotated in Fig. 3) and most others are extracellular matrix components, some of which are known to down-regulate TGFβ1 signaling, providing feedback loops [42–44]. Several others consist in extracellular matrix modifiers and degradation regulators, such as HTRA1, PCOC1, TIMP 1, which are likely to participate in TGFβ1-induced extracellular remodeling. The list of proteins described herein is relatively short, yet sufficient to evidence patient-to-patient differences (Supplemental data 3, 5, 6, 8) and metabolic changes in response to growth factors (Fig. 3). The responsiveness of primary human chondrocytes to TGFβ is known to be influenced by age [45] and inflammation [46]. However, basal secretion levels between donors have
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
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Patient 3 approx. TGF Ctrl TGF Ctrl TGF MW (kDa) 35
35
TF
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CPII
60
490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513
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489
R
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O
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N
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rarely been taken into account and have never been compared at a proteomic level. Noticeably, here, at both 48 and 96 hours post-stimulation, the vast majority of up-regulations were most pronounced for one donor (#2), while all three suffered from advanced osteoarthritis. Interestingly, the donor who exhibited the highest up-regulations also exhibited the lowest basal secretion levels of the corresponding proteins (Supplemental data 3). Therefore, inter-patient differential responsiveness to TGFβ1 might be due to differences in their basal metabolic/differentiation states, more directly than, for instance, endogenous levels of cytokines and their cognate receptors. Increasing efforts are being deployed towards qualitative and quantitative characterization of secretomes using mass spectrometry (174 Pubmed citations since 2013 out of 531 in total). An approach combining metabolic labeling and click chemistry was developed to allow secretome characterization from cells grown in the presence of serum [47]. However, whenever the response of a cell type to a given soluble mediator is investigated, the stimulation is in most cases performed under serum-free conditions, because serum components interfere with most stimuli. Also, in the expanding field of stem cell-based tissue repair, most protocols use serum-containing media for stem cell growth and pre-orientation, but serum-free media supplemented with minimal exogenous proteins for differentiation into mature cell types [1–3]. This is because serum-free medium formulations favor differentiation and resolve issues of serum compatibility with in vivo transplantation. Therefore, removing serum prior to secretome analysis is required for biological reasons. Under such circumstances, the conditioned medium
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Fig. 4 – Western blot analysis of the changes in secretion levels of individual proteins. Aliquots of the same culture media used in LC-MALDI at 96 hour time-point were precipitated and dissolved in loading buffer and directly analyzed. Abbreviated names of proteins are indicated on the left. CPI, C-propeptide of type Iα1 collagen; CPII, C-propeptide of type II collagen. Approximated molecular weights are indicated on the right. Transferrin (TF) was used as loading control as it originates from the culture medium.
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PGS2
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FN
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contains enriched secretome, facilitating downstream analysis. So far, proposed approaches for the quantitative analysis of serum-free secretomes require either metabolic labeling [5–8] or, to a lesser extent, isobaric labeling [9]. Although these methods proved efficient, metabolic labeling, as click chemistry, remains particularly expensive and can difficultly be considered as a non-invasive technique. Routine secretome characterization can only be achieved using sufficiently robust, rapid, straightforward and yet affordable sample preparation and mass spectrometry methods. The method developed here was designed for being as rapid and simple as possible. Trypsin digests were separated through short LC runs (1 hour in total) combined to moderate fractionation on MALDI plates (170 fractions). Quantitative acquisition required no more than 3 hours for a full 18-sample experiment with our instrument and yet results were statistically robust (Fig. 2). Then, of course, assigning fold-changes of compounds to peptides and summarizing them per protein required MS/MS, which is more long lasting if performed for each sample. However, the off-line nature of the LC-MALDI process, combined to the statistical tools described here, turns into a fundamental advantage as it allows to (i) generate exclusion and inclusion lists based on MS data which can then be used for MS/MS using the same LC separation and (ii) assess the reproducibility of LC runs based on MS data, which eventually allows assigning identities obtained through MS/ MS analysis of a series of runs to compounds observed in one or several others. In particular when analyzing series of biological samples which contain the same proteins in variable amounts, this results in a spectacular decrease of the machine time usage for identification. Therefore, the practical and theoretical workflow described herein (summarized in Fig. 1) is very well suited for routine comparisons. A major drawback of using short LC gradients and minimal fractionation is the poor depth of proteome coverage. However, in the context of, for instance, quality control of chondrogenic biomaterials or qualitative evaluation of chondrogenic stimuli to be applied to improve chondrogenesis, such depth of analysis will be sufficient as it covers molecules representative of the chondrocyte phenotype [48] and of its dedifferentiation [49]. When willing to establish a quantification method, the most recognized demonstration consists in spiking exogenous proteins at various known concentrations into the samples of interest and verifying that the method does evidence the expected fold changes. One limitation of our study is that, instead, we diluted secretome-containing medium into secretome-free medium, because it allowed validating the method over the very same protein identities and concentrations as those found in the biological samples. Since the total protein content of secretome-containing medium was about twice that of secretome-free medium (Supplemental data 1), the complexities of all samples were not identical and this could have resulted in significant deviations in the calculation of peptide/protein ratios. On another hand, secretomes are very dynamic populations (illustrated in Supplemental data 1) and therefore, methods to monitor their proteomic changes need to cope with such constraints; indeed, calculations were very close to expectations in our validation experiments (Fig. 2). In terms of quantification, the main limit of the label-free LC-MALDI workflow evidenced here was the relatively small
D
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Patient 2
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Patient 1
Please cite this article as: Riffault M, et al, Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation, J Prot (2014), http://dx.doi.org/10.1016/j.jprot.2014.10.026
514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573
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JOURNAL OF P ROTEOM IC S XX ( 2014) X XX–X XX
587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609
615 616 617 618
ammonium bicarbonate false discovery rate lack of discovery rate coefficient of variation
620 619
Author contributions
621 623
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
625 624
Funding sources
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This work was supported by Association pour la Recherche contre le Cancer, Ligue Régionale contre le Cancer, Fondation
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627
631 Q4 630
[29,30,31,33,34,35,36,37,38,39,40,41]
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Transparency document
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Uncited references
The Transparency document associated with this article can 635 be found, in the online version. 636
O
AB FDR LDR CV
579
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614
578
Acknowledgment
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The study was performed under the framework of Fédération 639 de Recherche 3209, Bioingénieurie Moléculaire, Cellulaire et 640 Thérapeutique. 641
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Abbreviations
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REFERENCES
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pour la Recherche Médicale, Région Lorraine, the Centre 628 Hospitalier Universitaire of Nancy. 629
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dynamic range within which calculations were allowed: foldchanges lower than 0.05 were hardly measurable (Supplemental data 2) and resulted in high CV values and relative errors (Fig. 2). However, precise measurements of drastic fold changes are rarely needed to answer biological questions. In contrast, whenever measuring fold changes close to 1, both CVs and relative errors fell down dramatically, allowing measurements of 1.5-fold changes with a theoretical FDR lower than 1%, which is very satisfactory. Altogether, these data demonstrate the efficacy of label-free differential LC-MALDI in superficial proteome differential analysis and its application to chondrocyte secretome investigations. Other authors have reported the feasibility of such approaches in other biological contexts [21] but it is to our knowledge the first combination of label-free LC-MALDI to secretome analysis. Determining whether it will also apply to other, more complex proteomes with adaptations will require farther investigations. The possibility to compare protein levels using label-free, MALDI-based methods offers major practical advantages: associated cell culture methods are rather simple and economically much more affordable than those requiring metabolic labeling. Also, mass spectrometry is very little time consuming in this workflow, resulting in much decreased analytical costs. Therefore, it is much more likely to be used for routine experiments than previous alternatives. Finally, it offers the advantages of label-free relative quantification approaches to those who have easy access only to MALDIbased mass spectrometry. As pointed by others who have performed label-free differential LC-MALDI before [21,50], the lack of adapted analysis softwares long remained a limitation in terms of raw data handling and compilation. The combination of softwares used here provides a profound improvement in this respect. Upcoming versions should be more user-friendly and better adapted to LC-MALDI driven processes to facilitate its dissemination. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2014.10.026.
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