Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip Ayse Nur Polat, Karsten Kraiczek, Albert J. R. Heck, Reinout Raijmakers & Shabaz Mohammed Analytical and Bioanalytical Chemistry ISSN 1618-2642 Volume 404 Number 8 Anal Bioanal Chem (2012) 404:2507-2512 DOI 10.1007/s00216-012-6395-7
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Author's personal copy Anal Bioanal Chem (2012) 404:2507–2512 DOI 10.1007/s00216-012-6395-7
TECHNICAL NOTE
Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip Ayse Nur Polat & Karsten Kraiczek & Albert J. R. Heck & Reinout Raijmakers & Shabaz Mohammed
Received: 7 June 2012 / Revised: 30 July 2012 / Accepted: 29 August 2012 / Published online: 14 September 2012 # Springer-Verlag 2012
Abstract Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO2) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible. Keywords HPLC chip . Microfluidics . Dimethyl labeling . Phosphorylation . Quantification . TiO2 Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6395-7) contains supplementary material, which is available to authorized users. A. N. Polat : A. J. R. Heck : R. Raijmakers : S. Mohammed (*) Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands e-mail:
[email protected] S. Mohammed e-mail:
[email protected] A. N. Polat : A. J. R. Heck : S. Mohammed Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands K. Kraiczek Agilent Technologies R&D and Marketing GmbH and Company KG, Hewlett-Packard-Strasse 8, 76337 Waldbronn, Germany
Introduction Protein phosphorylation is one of the most studied PTMs and helps control signaling pathways for many biological functions [1]. Phosphorylation is often a sub-stoichiometric process and due to its transient nature, observation is challenging. Therefore, highly sensitive methods are required for the detection, identification, and quantification of phosphorylation events [2]. Mechanistic aspects of regulation can be highlighted by the abundance of specific phosphorylation events and so the ability to quantitate those has become an intensely studied topic [3–6]. The most commonly used strategies to obtain quantitative information involve stable isotope labeling [3, 7]. We and others have previously shown that automated labeling and quantitation of peptides can be performed with an inexpensive and straightforward technique, dimethyl labeling [8–11]. Dimethyl labeling is based on reductive amination of the primary amine groups of lysine residues and peptide N-termini [11]. Although the procedure was introduced as an excellent and robust solution-based protocol, we incorporated the use of C18based solid phase extraction to introduce automation and improved levels of recovery [9]. The C18 cartridge can simply be the online trap in an nanoliter flow rate LC-MS system or a part of a multidimensional chromatographic strategy [8, 9, 12]. As mentioned above, it is necessary to perform enrichment on samples for in-depth LC-MS/MS analysis of phosphorylated peptides. Titania enrichment is based on the selective bidentate binding of phosphorylated peptides onto the TiO2 surface at acidic pH and elution at alkaline pH [13, 14]. Although TiO2 has a high affinity for phosphorylated peptides, reproducible binding to the TiO2 surface is affected by rate of sample introduction. Thus, slow and controlled flow rates are required to obtain high and reproducible efficiency of binding [15, 16]. Automation of the enrichment procedure minimizes the manual interference and abolishes handling steps. Previously, we reported that performing an automated online TiO2 enrichment using a
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microfluidic HPLC phosphochip allows better sensitivity and resolution of the analysis [17, 18]. In this study, we show that the use of an HPLC phosphochip can also be combined with automated labeling to facilitate the quantitative analysis of phosphorylated peptides, by integrating TiO2-based enrichment and stable-isotope labeling into one automated online quantitative procedure. Advantageously, this automated procedure further eliminates contamination and sample losses during sample preparation.
FA (buffer A) and analysis was performed by switching the trap to be in line with the analytical column and nanoflow pump. A linear gradient from 5 to 100 % solvent B at 200 nL/min was used to separate the peptides. For the HPLC phosphochip and for the standard analysis, a 45-min gradient was used. Solvents used for analytical HPLC were 0.1 % FA (solvent A) and 0.1 % FA/80 % ACN (solvent B) for both chips. A 180-min gradient was used for complex sample analysis. On-chip labeling
Material and methods Materials All chemicals are supplied from Sigma, Germany unless otherwise stated. Please see “Electronic supplementary material” (ESM) for detailed descriptions for cell culture, lysis, digestion, dimethyl reagents, strong cation separation, MS identification, and quantification. HPLC chip LC-MS/MS All LC-MS/MS experiments were performed using an Agilent 1200 series HPLC-Chip LC system connected to an Agilent 6520 Q-ToF mass spectrometer (Agilent, Santa Clara, CA) [18]. For regular LC-MS/MS analyses, a commercial HPLCChip (G4240-62005) was used containing a 4 mm, 40 nL Zorbax 300SB C18 (5 μm; Agilent, Waldbronn, Germany) reverse phase column (RP) and a 43 mm×75 μm Zorbax 300SB C18 analytical column. For phosphorylated peptide enrichment, an Agilent custom HPLC phosphochip was used, with a three-sectioned trap column design consisting of first a 100-nL Aqua C18 RP trapping column (5 μm; Phenomenex, Torrance, CA), a 45-nL TiO2 column (5 μm, 300A, Sachtopore NP, Sachtleben, Germany), and a second 100-nL Aqua C18 (5 μm; Phenomenex, Torrance, CA) RP column [17]. Trapping and washing of peptides was performed at 3 μL/min using 2 %
The first sample was loaded onto RP1 (the first trap column) in 10 % FA using 100 % solvent A and labeled by injection of 40 μL (light) dimethyl labeling reagent, followed by removing the excess labeling reagent using a 20 μL 5 % FA injection, all at 3 μL/min. The second sample was, subsequently, loaded onto the first RP trap column in 10 % FA using 100 % solvent A and labeled by injection of 40 μL (heavy) dimethyl labeling reagent, followed by removing the excess labeling reagent using a 20 μL 5 % FA injection, all at 3 μL/min. Enrichment of phosphopeptides using the TiO2 column and simultaneous analysis of non–phosphopeptides was performed by switching the trap to be in line with the analytical column and nanoflow pump. A linear gradient from 5 to 100 % solvent B at 200 nL/ min was used to separate the peptides. This analysis was followed by injection of 2×20 μL elution buffer (250 mM NH4HCO3 pH9; 5 mM KF, 10 mM NaH2PO4, 1 mM Na2VO4) to elute the phosphorylated peptides from the TiO2 column to the (second trap column) RP2 column. Analysis of the phosphopeptides was performed by switching the RP2 column to be in line with the analytical column for a second linear gradient of a length appropriate to the complexity of the sample.
Results The aim of this study was to develop and evaluate an HPLC chip-based method for automated online phosphorylated
Table 1 Description of valve positions and location of both phosphorylated and regular peptides for each step of the procedure during labeling and enrichment on the HPLC phosphochip
1 2 3 4 5 6 7 8 9
Sample 1 loading Sample light labeling Wash Sample 2 loading Sample Heavy labeling Wash 1st gradient and enrichment Elution from TiO2 column 1 2nd gradient
Valve position
Non-phosphorylated peptides
Phosphorylated peptides
Loading Loading Loading Loading Loading Loading Analysis Loading Analysis
Reverse phase column Reverse phase column Reverse phase column Reverse phase column Reverse phase column Reverse phase column Analytical column – –
Reverse phase column Reverse phase column Reverse phase column Reverse phase column Reverse phase column Reverse phase column TiO2 column Reverse phase column Analytical column
1 1 1 1 1 1
1 1 1 1 1 1 2
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To be able to judge the automated procedure properly, we first set out to determine the sensitivity and linearity of our instrumental setup for phosphorylated peptides in a range of sample amounts typical for proteomics experiments. To this end, different amounts (50–1,000 fmol) of a tryptically
digested bovine alpha casein, beta casein, and albumin “standard” mixture were analyzed by LC-MS/MS on a regular C18 HPLC chip connected to an Agilent 6520 QToF mass spectrometer. We found that all the peptides have a good linear correlation, with regression coefficients (R2) close to 0.99, for the range tested (ESM, Fig. S1). In order to test the effect of the on-chip labeling on the behavior of the phosphorylated peptides, the dimethyl labeling was first performed on the standard C18 HPLC chip, as this has a more simple configuration (ESM, Fig. S2) compared to the HPLC phosphochip. Labeling on the HPLC chip is a straightforward procedure (ESM, Table S1). Based on previous studies, suitable triethylammonium bicarbonate
Fig. 1 Schematic of the HPLC phosphochip showing the trap and analytical column sections alongside active (pink) and passive flow paths Active parts and flow direction are indicated using pink. The steps required to label and enrich a single sample is shown. a Sample is loaded on to the first reverse phase column (RP1). b Sample bound to RP1 is labeled and subsequently the system is cleaned with a wash buffer. c The trap columns are then placed in line with analytical column and a gradient is applied, labeled phosphorylated peptides bind to TiO2 column and the regular peptides continue on to the analytical
column where they are separated (flowthrough). d Flow patch is once again changed and the trap eluent is sent to waste. An alkaline solution is introduced into the traps and the labeled phosphorylated peptides are eluted from TiO2 column and are bound to second reverse phase column (RP2) e Trap columns are then placed in line with analytical column again and a gradient is applied, the labeled phosphorylated peptides are then separated on the analytical column (elution). For a quantitative experiment steps, a and b are repeated for both samples and each uses labeling reagents with a unique isotopic composition
peptide enrichment and quantification. Since the ideal conditions for labeling and enrichment are different, optimization of reaction conditions was essential to perform a combined procedure. The reactivity of TiO2 with the labeling reagents had to be also considered. Isotope labeling on the HPLC chip
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(TEAB), sodium phosphate, and sodium acetate buffers were tested in order to obtain optimal reaction conditions for dimethyl labeling [8]. While testing the TEAB buffer, we observed contamination peaks hindering, the observation of peptides during an LC-MS analysis. No such issues were observed with the other two buffers where both sets produced “clean” chromatograms (ESM, Fig. S2). We found that the acetate buffer yielded marginally incomplete labeling and, therefore, the phosphate buffer was chosen as the optimal dimethyl labeling buffer. Integration of labeling and enrichment on HPLC phosphochip Automated online quantification of phosphorylated peptides requires the integration of online dimethyl labeling and TiO2 enrichment. Tables S1 and S2 (ESM) summarize the separate labeling and enrichment procedures. Table 1 contains the integrated labeling and enrichment procedure, where the two samples that will be quantitatively compared are referred to as “sample 1” and “sample 2”. A graphical representation of the integrated procedure is shown in Fig. 1. The compatibility of TiO2 enrichment with the procedures for online dimethyl labeling is critical, as reliable quantitative information can only be obtained when complete labeling and consistent enrichment is achieved. It should be noted that testing with differing amounts of peptide material established an upper limit of approximately 1 μg per labeling step. The integrated automated online quantification and phosphorylated peptide enrichment was evaluated by double labeling and analyzing 100 fmol of the standard mixture, i.e., both samples 1 and 2 consisted of this standard mixture. The total time required for the automated double labeling and enrichment was 215 min, when two 45-min analytical runs were performed. Mascot analyses of the LC/MS/MS data revealed no evidence of partial labeling or non-labeling of peptides. Typical base peak chromatograms of two independent experiments are shown in Fig. 2. All identified phosphorylated peptides and selected regular peptides were manually quantified using the areas of the extract ion chromatograms. As expected, the labeling ratio of 1:1 was confirmed by analyzing data through Spectrum Mill software. The inset in Fig. S4 (ESM) shows a typical result for a 1:1 phosphopeptide, which corresponds to a spectrum generated by integrating over the chromatographic peak. All our data clearly show that the enrichment efficiency and binding specificity of the HPLC phosphochip are not affected by the integration of the automated dimethyl labeling into the procedure. Furthermore, the ionization of peptides and spray stability were not compromised by any residual labeling reagent and a normal signal to noise ratio was observed during both analyses. Such a result is not surprising since none of the enrichment or labeling reagents are transferred
Fig. 2 Analysis of the standard mixture with the HPLC phosphochip where both enrichment and labeling is performed. a Base peak chromatograms of the elution b and flowthrough c of a double stable isotope dimethyl labeled 100 fmol sample. Replicate 1 is in purple and replicate 2 is in orange. Typical result (inset) for a 1:1 phosphopeptide (VPQLEIVPNpSAEER) which corresponds to a spectrum generated by integrating over the chromatographic peak
to the analytical column but are directed to waste. All peptides identified in the unlabeled online enrichment analysis were also identified in the automated online quantification analysis. We then repeated the labeling/enrichment procedures using differing ratios between sample 1 and 2. Ratios of 0.1:1 to 1:1 were tested. We found good linearity (R2 of 0.9963) between the expected and observed ratios (ESM, Fig. S4) demonstrating that differential phosphorylation can be accurately determined up to 10-fold increase or decrease of phosphorylation. Complex sample analysis We further tested the efficacy of our automated online quantification approach with a “real-life” complex sample, a tryptic digest of HeLa cells. We chose to use a sample that would be a fractionated sample and a fraction of which would contain a reasonable level of phosphorylated peptides. It has been shown that a low pH SCX separation can be used to generate a crude enrichment of phosphorylated
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peptides alongside fractionation of the whole lysate [19, 20]. We fractionated a HeLa lysate digestion and chose to analyze an SCX fraction that was partially enriched in phosphopeptides. Similar levels of enrichment were observed to those previously demonstrated for this chip [18] without the additional labeling step (ESM, Table S3). The resulting data were analyzed with Spectrum Mill which generated a mean log2 light/heavy ratio of −0.03 for all quantified peptides and phosphopeptides (ESM, Figs. S5 and S6). The plot of the ratios obtained in the eluted peptides is gratifyingly similar to what is achieved when labeling is performed “offline” [8].
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phosphorylated peptides over at least one order of magnitude and that the method is applicable to complex samples if pre-fractionation is performed. The main advantage of automated online quantification is the displacing of time spent performing several tasks from human to the HPLC system. Acknowledgments This work was in parts supported by the PRIMEXS project, grant agreement number 262067, funded by the European Union 7th Framework Programme; The Netherlands Proteomics Centre, embedded in the Netherlands Genomics Initiative, The Netherlands Organization for Scientific Research (NWO) with the VIDI Grant 700.10.429 for SM; and with a grant from the Agilent Technologies Foundation.
Discussion
References
Here, we developed an integrated automated online labeling and enrichment procedure using the HPLC phosphochip. Not unexpectedly, the phosphate buffer system was found to be optimal for the HPLC phosphochip [9]. Although we anticipated that the use of a phosphate buffer system might interfere with the HPLC phosphochip, we did not find any compromise of the enrichment efficiency of phosphorylated peptides. Differences between isotopologues in terms of retention time (due to the incorporation of deuterium) were not particularly observable in the HPLC chip system. The combined online labeling and enrichment approach developed in this study also minimizes sample loss and systematic errors and diminishes complications often occurring during chemical labeling. Still, several other parameters can influence the identification and the quantification of phosphorylated peptides including the low abundance of many phosphorylated peptides, meaning it is crucial to have a well-defined and robust approach [21]. In our complex sample analysis, we observed a broader ratio distribution for the quantified peptides in the flow-through (ESM, Fig. S6) when compared to the elution analysis (ESM, Fig. S5). This is caused by the relation between precision and peptide intensity. In our case, the intensity of regular peptides is lower in this SCX fraction when compared to phosphopeptides. An optimized robust automated online quantification system such as developed here can be beneficial for phosphoproteome analysis when only small sample amounts are available (up to 1 μg) since the method requires less time, expertise and labor, and is less prone to error and sample losses during the whole procedure.
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Conclusion We demonstrate that it is feasible to perform both labeling and phosphopeptide enrichment using a microfluidic device. We show good linearity of integrated EIC areas for several
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