Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics
2017; Elsevier BV; Volume: 16; Issue: 4 Linguagem: Inglês
10.1074/mcp.o116.065136
ISSN1535-9484
AutoresNils A. Kulak, Philipp E. Geyer, Matthias Mann,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoRecent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed "spider fractionator" in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2–96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements. Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed "spider fractionator" in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2–96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements. Mass spectrometry (MS)-based bottom-up proteomic workflows consist of multiple steps, namely sample preparation, on-line liquid chromatography (LC) coupled with MS measurements, followed by computational data analysis, and interpretation. LC-MS/MS technologies have improved drastically from the initial identification of one or a few proteins using manual, complex, and time-consuming protocols to essentially complete proteomic coverage of microorganisms in a rapid and streamlined manner (1.Beck M. Claassen M. Aebersold R. Comprehensive proteomics.Curr. Opin. Biotechnol. 2011; 22: 3-8Crossref PubMed Scopus (72) Google Scholar, 2.Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The one hour yeast proteome.Mol. Cell. Proteomics. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 3.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. 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Therefore, there is a continuing need for more powerful workflows, and here we contribute to these efforts in the important area of peptide pre-separation before the LC-MS/MS analysis. To yield in-depth proteomes of complex biological samples, two-dimensional separation approaches at the peptide level are attractive because they are more universally applicable than protein level fractionation. First dimension separation techniques range from isoelectric focusing (6.Hörth P. Miller C.A. Preckel T. Wenz C. Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis.Mol. Cell. Proteomics. 2006; 5: 1968-1974Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 7.Essader A.S. Cargile B.J. Bundy J.L. Stephenson Jr., J.L. 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High pH reversed-phase fractionation, alternatively termed basic reversed-phase, as a first off-line chromatography separation in combination with the low pH reversed-phase fractionation in the second on-line dimension was first demonstrated more than 10 years ago. In comparison with other methodologies, it benefits from the uniform first dimensional peptide elution profiles achievable with high pH reversed-phase separation and the high peptide separation efficiency in both dimensions (18.Delmotte N. Lasaosa M. Tholey A. Heinzle E. Huber C.G. Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis.J. Proteome Res. 2007; 6: 4363-4373Crossref PubMed Scopus (117) Google Scholar, 19.Manadas B. English J.A. Wynne K.J. Cotter D.R. Dunn M.J. Comparative analysis of OFFGel strong cation exchange with pH gradient, and RP at high pH for first-dimensional separation of peptides from a membrane-enriched protein fraction.Proteomics. 2009; 9: 5194-5198Crossref PubMed Scopus (62) Google Scholar). Because the two separation dimensions are not completely orthogonal (meaning that peptide retention times are still correlated), direct application of high pH fractionation would lead to non-uniform filling of the gradient in the second dimension. The key advance that solved this problem was the combination of fractions that elute at substantially different times in the first dimension (13.Gilar M. Olivova P. Daly A.E. Gebler J.C. Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions.J. Sep. Sci. 2005; 28: 1694-1703Crossref PubMed Scopus (375) Google Scholar). This "concatenation" tends to uniformly fill the gradients, leading to deeper proteomes independent of the nature of the sample, while maintaining throughput (20.Wang Y. Yang F. Gritsenko M.A. Wang Y. Clauss T. Liu T. Shen Y. Monroe M.E. Lopez-Ferrer D. Reno T. Moore R.J. Klemke R.L. Camp 2nd, D.G. Smith R.D. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.Proteomics. 2011; 11: 2019-2026Crossref PubMed Scopus (403) Google Scholar, 21.Dwivedi R.C. Spicer V. Harder M. Antonovici M. Ens W. Standing K.G. Wilkins J.A. Krokhin O.V. Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics.Anal. Chem. 2008; 80: 7036-7042Crossref PubMed Scopus (138) Google Scholar, 22.Song C. Ye M. Han G. Jiang X. Wang F. Yu Z. Chen R. Zou H. 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Offline high pH reversed-phase peptide fractionation for deep phosphoproteome coverage.Methods Mol. Biol. 2016; 1355: 179-192Crossref PubMed Scopus (33) Google Scholar). Despite the success of high pH reversed-phase fractionation in the deep characterization of complex proteomic samples, a current limitation is the requirement for rather large amounts of starting material. This is due to the large column diameters, flow rates, and number of fractions that are collected before concatenation to preserve peak separation from the first dimension and to maintain collection volumes that can easily be handled. Therefore, instead of the nanoflow systems typical of on-line separation, much larger columns and flow rates are almost always employed. This in turn requires large sample sizes, and milligram amounts of starting material are typical for high pH reversed-phase fractionation. Unfortunately, this implies high reagent costs, for instance for proteolytic enzymes or for the chemical labeling reagents used in multiplexing. Furthermore, it restricts deep proteomes preliminary to cases where comparatively large protein amounts are available and excludes the investigation of rare cellular subpopulation or laser micro-dissected cells in tumor tissues, for instance. High pH fractionation with higher flow rates and larger sample amounts is also used to investigate post-translational modifications in great depth by the combination of isobaric mass tag labeling of peptides after digestion, followed by high pH fractionation and consecutive enrichments (28.Mertins P. Yang F. Liu T. Mani D.R. Petyuk V.A. Gillette M.A. Clauser K.R. Qiao J.W. Gritsenko M.A. Moore R.J. Levine D.A. Townsend R. Erdmann-Gilmore P. Snider J.E. Davies S.R. et al.Ischemia in tumors induces early and sustained phosphorylation changes in stress kinase pathways but does not affect global protein levels.Mol. Cell. Proteomics. 2014; 13: 1690-1704Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). In such cases, large sample amounts and high volumes are necessary because of the subsequent enrichment. However, post-translational modification analysis could benefit from a drastic scale-down in high pH fractionation, if already enriched peptides are fractionated. This would necessitate high sensitivity of the fractionation step and be economically attractive in terms of labeling reagents. Here, we describe a novel approach that allows efficient sample concatenation without using large volumes. Instead, nanoflow systems are employed, and the intermediate sample collection step is eliminated. We demonstrate the operating principle of our spider fractionator, show that fractionation efficiency remains very high, and establish that the flexibility of the system allows choosing an optimum balance between measurement time and desired depth of proteome coverage. Very low sample amounts can be separated without apparent fractionation-induced sample losses. We demonstrate the sensitivity of the system by the analysis of 12 human cell lines to a depth of about 10,000 proteins with only 1 μg of sample. HeLa cells were cultured in high glucose DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin (all from Life Technologies, Inc.). Cell lines were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% dialyzed fetal bovine serum and penicillin/streptomycin. Cells were counted using a countess cell counter (Invitrogen), and aliquots of 106 cells were snap-frozen and stored at −80 °C. Protein concentrations were determined after solubilizing the samples in 8 m urea by tryptophan fluorescence emission at 350 nm using an excitation wavelength of 295 nm. Tryptophan at a concentration of 0.1 μg/μl in 8 m urea was used to establish a standard calibration curve (0–4 μl). From this, we estimated that 0.1 μg/μl tryptophan are equivalent to the emission of 7 μg/μl of human protein extract, assuming that tryptophan on average accounts for 1.3% of human protein amino acid composition. Sample preparation was performed as described previously (3.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar) with the following adaptations. 300 μg of cells were suspended in 50 μl of SDC reduction and alkylation buffer (3.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar). We used 2-chloro-N,N-diethylacetamide as alkylating reagent for the comparison of the 13 cell lines and 2-chloro-acetamide for all other experiments. The cells were kept at 95 °C for 5 min to denature proteins and afterward sonicated to shear DNA and enhance cell disruption with a water bath sonicator (Bioruptor, model UCD-200, Diagenode) for 15 min at the maximum level. The proteolytic enzymes LysC and trypsin were added in a 1:100 ratio (micrograms of enzyme to micrograms of protein), and the solution was incubated for 4 h at 37 °C. Peptides were acidified by adding 100 μl of ethyl acetate, 1% TFA and extensive mixing for 2 min, and 20 μg were transferred into StageTips containing two 14-gauge SDB-RPS (poly(styrene-divinylbenzene) reversed phase sulfonate) plugs. Afterward, the StageTips were washed with 100 μl of ethyl acetate, 1% TFA to strip SDC and lipids from the digested cells. This was followed by a wash step with 100 μl of ddH2O 1The abbreviations used are: ddH2O, double distilled H2O; SMC, smooth muscle cell; EC, embryonic carcinoma., 0.2% TFA. The purified peptides were eluted with 60 μl of 80% acetonitrile, 19% ddH2O, 1% ammonia in autosampler vials. For all steps, the StageTips were centrifuged at 2,000 × g until the solutions were rinsed through completely. The collected material was dried using a SpeedVac centrifuge at 60 °C (Eppendorf, Concentrator Plus). Peptides were suspended in 2% acetonitrile, 0.1% TFA in ddH2O and sonicated for 15 min in a water bath sonicator (Branson Ultrasonics, Ultrasonic Cleaner Model 2510). Moreover, 6,600 HeLa cells, the equivalent to 1 μg of starting material (29.Finka A. Goloubinoff P. Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.Cell Stress Chaperones. 2013; 18: 591-605Crossref PubMed Scopus (143) Google Scholar), were separately digested using the in-StageTip protocol (3.Kulak N.A. Pichler G. Paron I. Nagaraj N. Mann M. Minimal encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref PubMed Scopus (1002) Google Scholar) with the above mentioned adaptations. We constructed a software-controlled, fully automated, rotor-valve-based fraction collector system coupled on line to a nanoflow HPLC (EASY-nLC 1000 system, Thermo Fisher Scientific), and we used this for all high pH reversed-phase pre-fractionations. The fraction collector system was named Spider Fractionator and is under commercial development by PreOmics GmbH, Martinsried, Germany. We provide a list of components used in constructing the fractionator (supplemental Table 1). For the work reported here, we standardized on a first dimension column of 250 μm inner diameter and a length of 30 cm, which was packed with 1.9 μm C18 particles (ReproSil-Pur C18-AQ 1.9 μm resin by Dr. Maisch GmbH) and has an estimated loading capacity of at least 100 μg. The column is available from PreOmics GmbH (Article No. P.O. 00007). All columns (first dimension and on-line dimension) were passivated by a single run of BSA to saturate irreversible binding sites. For separation into eight pooled fractions, we loaded 20 μg (or other amounts where indicated) of purified and digested peptides onto a reversed-phase C18 column. A gradient was generated by using a dual buffer system (buffers A and B) also from PreOmics GmbH (Article No. P.O. 00009). Peptides were eluted from 3% B to 30% B in 45 min followed by a linear increase to 60% B in 17 min. This gradient was followed by a further linear increase to 95% B in 5 min and a 3 min wash at 95% B, followed by a 10 min decrease to 3% B. The last segments ensure that the output lines (volume about 800 nl) are emptied, and none of the remaining peptides are lost. The flow rate was kept at a constant 2 μl/min. The 96-well plate was moved by a stepper motor-driven linear actuator. Software was implemented on a Raspberry microcontroller. We separated peptides into 4, 8, 16, and 24 fractions using rotor valve shifts of 90 s. Fractions were collected into 0.2-ml thin-walled 8-tube strips (Thermo Fisher Scientific). We loaded 20 μg of starting material for 4 and 8 fractions, 40 μg for 16 fractions, and 60 μg for 24 fractions. The concatenation scheme of table I was used for pooling. For a more detailed version of the fractionation schemes see supplemental Fig. 1.Table IConcatenation schemeNo. pooled fractions481624Peptide amount (μg)20204060No. of non-pooled fractions54545454Pooling scheme1;5;9;13;17;21;25;29;33;37;41;45;49;531;9;17;25;33;41;491;17;33;491;25;492;6;10;14;18;22;26;30;34;38;42;46;50;54; etc.2;10;18;26;34;42;50; etc.2;18;34;50; etc.2;26;50; etc. Open table in a new tab The pooled fractions were dried using a SpeedVac centrifuge at 60 °C (Eppendorf, Concentrator Plus). Peptides were suspended in 2% acetonitrile, 0.1% TFA in ddH2O and sonicated for 15 min in a water bath sonicator (Branson Ultrasonics, Ultrasonic Cleaner Model 2510). A total of 2 μg of each concatenated fraction was loaded and measured by LC-MS/MS as described below. We used the same buffers, gradients, and pooling scheme as for the spider fractionator system in comparison with a higher flow system and to a recently introduced spin column system. For all three systems, the same HeLa digest was used to fractionate 1 or 20 μg of peptides. The higher flow system consisted of an XBridge peptide BEH C18 column (2.5 μm particle size, 2.1 × 250 mm, Waters) with a Shimadzu HPLC system at a 60 °C run at a flow rate of 150 μl/min. The fractions were manually pooled. For the 1 μg sample, all fractions were re-pooled into a single vial to determine sample loss. For the 20 μg sample, we manually concatenated samples according to the same scheme as automatically done by the spider fractionator. On the spin system (high pH reversed-phase peptide fractionation kit, Pierce catalog number 84868), separation was done according to the manufacturer's instructions resulting in eight fractions but no concatenation. Samples were measured using LC-MS instrumentation consisting of an EASY-nLC 1000 ultra-high pressure system (Thermo Fisher Scientific) coupled via a nanoelectrospray ion source (Thermo Fisher Scientific) to a hybrid quadrupole Orbitrap mass spectrometer (Q Exactive HF Orbitrap from Thermo Fisher Scientific) (30.Scheltema R.A. Hauschild J.P. Lange O. Hornburg D. Denisov E. Damoc E. Kuehn A. Makarov A. Mann M. The QExactive HF, a Benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultra-high-field Orbitrap analyzer.Mol. Cell. Proteomics. 2014; 13: 3698-3708Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 31.Kelstrup C.D. Jersie-Christensen R.R. Batth T.S. Arrey T.N. Kuehn A. Kellmann M. Olsen J.V. Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.J. Proteome Res. 2014; 13: 6187-6195Crossref PubMed Scopus (137) Google Scholar). Purified peptides were separated on 40 cm HPLC columns (75 μm inner diameter; in-house packed into the tip) at 60 °C with ReproSil-Pur C18-AQ 1.9 μm resin by Dr. Maisch GmbH). For all measurements, peptides were loaded in buffer A (0.1% formic acid, 5% DMSO (32.Hahne H. Pachl F. Ruprecht B. Maier S.K. Klaeger S. Helm D. Médard G. Wilm M. Lemeer S. Kuster B. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.Nat. Methods. 2013; 10: 989-991Crossref PubMed Scopus (176) Google Scholar)) and eluted with a linear 55 min gradient of 2–20% of buffer B (0.1% formic acid, 5% DMSO, 80% acetonitrile), followed by an increase to 40% buffer B within 40 min and afterward within 2 min to 98% buffer B and a 2 min wash at 98% buffer B. The flow rate was kept at 350 nl/min. Column temperature was kept at 60 °C by an in-house-developed oven containing a Peltier element, and parameters were monitored in real time by the SprayQC software (33.Scheltema R.A. Mann M. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components.J. Proteome Res. 2012; 11: 3458-3466Crossref PubMed Scopus (44) Google Scholar). MS data was acquired with the Thermo Xcalibur software version 3.0.63, a topN method where N could be up to 100. This method in principle allows a very large number of precursor peaks to be picked for fragmentation but is in practice limited by the number of precursors with sufficient ion intensity. In the entire data set, N was 15 on average. Target values for the full scan MS spectra were 3 × 106 charges in the 300–1,650 m/z range with a maximum injection time of 15 ms. Transient times corresponding to a resolution of 60,000 at m/z 200 were chosen. A 1.5 m/z isolation window and a fixed first mass of 100 m/z were used for MS/MS scans. Fragmentation of precursor ions was performed by higher energy C-trap dissociation (34.Olsen J.V. Macek B. Lange O. Makarov A. Horning S. Mann M. Higher-energy C-trap dissociation for peptide modification analysis.Nat. Methods. 2007; 4: 709-712Crossref PubMed Scopus (720) Google Scholar) with a normalized collision energy of 27 eV. MS/MS scans were performed at a resolution of 15,000 at m/z 200 with an ion target value of 5 × 104 and a maximum injection time of 25 ms. Dynamic exclusion was set to 30 s to avoid repeated sequencing of identical peptides. MS raw data files were analyzed by MaxQuant software version 1.5.3.31 (35.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar), and peptide lists were searched by the Andromeda search engine (36.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3474) Google Scholar) against the human Uniprot FASTA database to which common contaminant proteins had been added (86,746 entries) with cysteine diethylcarbamidomethylation as a fixed modification for the comparison of the 13 cell lines and cysteine carbamidomethylation as a fixed modification for all other experiments. N-terminal acetylation and methionine oxidations were used as variable modifications in all experiments. The false discovery rate was set to 0.01 for both proteins and peptides with a minimum length of 7 amino acids and was determined by searching a reverse database. Enzyme specificity was set to trypsin and a maximum of two missed cleavages were allowed in the database search. Peptide identification were performed with an allowed initial precursor mass deviation up to 7 ppm and an allowed fragment mass deviation of 20 ppm. The MaxQuant feature "match between runs" was enabled within the dataset of the pooled eight fractions and the single shot samples for all cell line samples. Proteins matching the reversed database were filtered out. Label-free protein quantification was done with a minimum ratio count of 1 (37.Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2715) Google Scholar). All bioinformatics analyses were performed within the Perseus software of the MaxQuant computational platform (35.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biot
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