An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*
2015; Elsevier BV; Volume: 14; Issue: 6 Linguagem: Inglês
10.1074/mcp.m114.045724
ISSN1535-9484
AutoresSiyang Li, Brian D. Plouffe, Arseniy M. Belov, Somak Ray, Xianzhe Wang, Shashi K. Murthy, Barry L. Karger, Alexander R. Ivanov,
Tópico(s)Single-cell and spatial transcriptomics
ResumoIsolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. Rare cells in blood and tissue have been shown to serve as specific indicators of disease status and progression, a source of adult stem cells, and a tool for patient stratification and monitoring. Previous reports (1Nagrath S. Sequist L.V. Maheswaran S. Bell D.W. Irimia D. Ulkus L. Smith M.R. Kwak E.L. Digumarthy S. Muzikansky A. Ryan P. Balis U.J. Tompkins R.G. Haber D.A. Toner M. Isolation of rare circulating tumor cells in cancer patients by microchip technology.Nature. 2007; 450: 1235-1239Crossref PubMed Scopus (2963) Google Scholar, 2Cristofanilli M. Budd G.T. Ellis M.J. Stopeck A. Matera J. Miller M.C. Reuben J.M. Doyle G.V. Allard W.J. Terstappen L.W. Hayes D.F. Circulating tumor cells, disease progression, and survival in metastatic breast cancer.New Engl. J. Med. 2004; 351: 781-791Crossref PubMed Scopus (3637) Google Scholar, 3Gleghorn J.P. Pratt E.D. Denning D. Liu H. Bander N.H. Tagawa S.T. Nanus D.M. Giannakakou P.A. Kirby B.J. Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody.Lab Chip. 2010; 10: 27-29Crossref PubMed Scopus (314) Google Scholar, 4Riethdorf S. Fritsche H. Müller V. Rau T. Schindlbeck C. Rack B. Janni W. Coith C. Beck K. Jänicke F. Jackson S. Gornet T. Cristofanilli M. Pantel K. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cellsearch system.Clin. Cancer Res. 2007; 13: 920-928Crossref PubMed Scopus (1075) Google Scholar), for example, have shown that the concentration of circulating tumor cells (CTCs) within a cancer patient's blood can act as a therapeutic monitoring tool (1Nagrath S. Sequist L.V. Maheswaran S. Bell D.W. Irimia D. Ulkus L. Smith M.R. Kwak E.L. Digumarthy S. Muzikansky A. Ryan P. Balis U.J. Tompkins R.G. Haber D.A. Toner M. Isolation of rare circulating tumor cells in cancer patients by microchip technology.Nature. 2007; 450: 1235-1239Crossref PubMed Scopus (2963) Google Scholar, 2Cristofanilli M. Budd G.T. Ellis M.J. Stopeck A. Matera J. Miller M.C. Reuben J.M. Doyle G.V. Allard W.J. Terstappen L.W. Hayes D.F. Circulating tumor cells, disease progression, and survival in metastatic breast cancer.New Engl. J. Med. 2004; 351: 781-791Crossref PubMed Scopus (3637) Google Scholar, 3Gleghorn J.P. Pratt E.D. Denning D. Liu H. Bander N.H. Tagawa S.T. Nanus D.M. Giannakakou P.A. Kirby B.J. Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody.Lab Chip. 2010; 10: 27-29Crossref PubMed Scopus (314) Google Scholar, 4Riethdorf S. Fritsche H. Müller V. Rau T. Schindlbeck C. Rack B. Janni W. Coith C. Beck K. Jänicke F. Jackson S. Gornet T. Cristofanilli M. Pantel K. Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cellsearch system.Clin. Cancer Res. 2007; 13: 920-928Crossref PubMed Scopus (1075) Google Scholar). Additionally, the isolation of adult stem cells provides a needed cell source for tissue engineering and regenerative medicine treatments (5Green J.V. Radisic M. Murthy S.K. Deterministic lateral displacement as a means to enrich large cells for tissue engineering.Anal. Chem. 2009; 81: 9178-9182Crossref PubMed Scopus (78) Google Scholar, 6Plouffe B.D. Kniazeva T. Mayer Jr., J.E. Murthy S.K. Sales V.L. Development of microfluidics as endothelial progenitor cell capture technology for cardiovascular tissue engineering and diagnostic medicine.FASEB J. 2009; 23: 3309-3314Crossref PubMed Scopus (81) Google Scholar). Finally, separation and genomic analysis of key cell populations from patients allows for targeted treatment regimens (7Hamburg M.A. Collins F.S. The path to personalized medicine.New Engl. J. Med. 2010; 363: 301-304Crossref PubMed Scopus (1376) Google Scholar, 8Yu M. Bardia A. Wittner B.S. Stott S.L. Smas M.E. Ting D.T. Isakoff S.J. Ciciliano J.C. Wells M.N. Shah A.M. Concannon K.F. Donaldson M.C. Sequist L.V. Brachtel E. Sgroi D. Baselga J. Ramaswamy S. Toner M. Haber D.A. Maheswaran S. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition.Science. 2013; 339: 580-584Crossref PubMed Scopus (1816) Google Scholar). Rare cells in blood or other body fluids represent a particularly challenging problem for discovery proteomic analysis as the volume of the fluid sample is limited and the concentration of cells within that sample is very low. For a blood sample containing rare cells of interest, this low level means capturing a subpopulation of target cells with high recovery and purity from a greatly heterogeneous mixture in only one or a few ml and then performing sample preparation with minimal sample loss. Furthermore, ultra-trace LC-MS needs to be conducted with specially prepared columns with highly sensitive MS, along with advanced data processing. Key to success is the full integration of all the steps in the workflow to achieve the detection level required. The present work combines a series of innovative steps leading to successful discovery proteomic analysis of rare cells. Consider first rare cell isolation for which several approaches have recently been developed (9Pratt E.D. Huang C. Hawkins B.G. Gleghorn J.P. Kirby B.J. Rare cell capture in microfluidic devices.Chem. Eng. Sci. 2011; 66: 1508-1522Crossref PubMed Scopus (161) Google Scholar, 10Zborowski M. Chalmers J.J. Rare cell separation and analysis by magnetic sorting.Anal. Chem. 2011; 83: 8050-8056Crossref PubMed Scopus (147) Google Scholar). A particularly powerful approach is magnet-activated cell sorting (MACS) where antibody-functionalized magnetic beads are utilized to enrich a subset of cells in a complex sample such as whole blood (10Zborowski M. Chalmers J.J. Rare cell separation and analysis by magnetic sorting.Anal. Chem. 2011; 83: 8050-8056Crossref PubMed Scopus (147) Google Scholar, 11Miltenyi S. Müller W. Weichel W. Radbruch A. High gradient magnetic cell separation with MACS.Cytometry. 1990; 11: 231-238Crossref PubMed Scopus (1446) Google Scholar). Although magnet-activated cell sorting-based and other microfluidic approaches of cell separation have recently shown the ability to isolate rare cells (e.g. 90%) and efficiency (>95%)(12Plouffe B.D. Mahalanabis M. Lewis L.H. Klapperich C.M. Murthy S.K. Clinically relevant microfluidic magnetophoretic isolation of rare-cell populations for diagnostic and therapeutic monitoring applications.Anal. Chem. 2012; 84: 1336-1344Crossref PubMed Scopus (81) Google Scholar, 13Ozkumur E. Shah A.M. Ciciliano J.C. Emmink B.L. Miyamoto D.T. Brachtel E. Yu M. Chen P.I. Morgan B. Trautwein J. Kimura A. Sengupta S. Stott S.L. Karabacak N.M. Barber T.A. Walsh J.R. Smith K. Spuhler P.S. Sullivan J.P. Lee R.J. Ting D.T. Luo X. Shaw A.T. Bardia A. Sequist L.V. Louis D.N. Maheswaran S. Kapur R. Haber D.A. Toner M. Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells.Science Trans. Med. 2013; 5: 179ra147Crossref Scopus (779) Google Scholar, 14Karabacak N.M. Spuhler P.S. Fachin F. Lim E.J. Pai V. Ozkumur E. Martel J.M. Kojic N. Smith K. Chen P.-i. Yang J. Hwang H. Morgan B. Trautwein J. Barber T.A. Stott S.L. Maheswaran S. Kapur R. Haber D.A. Toner M. Microfluidic, marker-free isolation of circulating tumor cells from blood samples.Nat. Protocols. 2014; 9: 694-710Crossref PubMed Scopus (549) Google Scholar), the potential of these systems in enabling downstream molecular analyses has yet to be fully realized. Microfluidic channels, in comparison to traditional magnet-activated cell sorting, allow for improved control of the magnetic field for precise focusing in the microchannels, resulting in higher efficiency, recovery, and purity of isolation. For proteomic analysis, rare cell isolation is followed by a series of sample preparation steps, for example cell lysis and protein extraction and digestion. Several approaches such as denaturant-assisted lysis, acetone precipitation, filter-aided sample preparation, and monolithic microreactor-based techniques have been developed for processing small amounts of sample, for example 500–1000 cultured cells (15Wang N. Xu M. Wang P. Li L. Development of mass spectrometry-based shotgun method for proteome analysis of 500 to 5000 cancer cells.Anal. Chem. 2010; 82: 2262-2271Crossref PubMed Scopus (71) Google Scholar, 16Tian R. Wang S. Elisma F. Li L. Zhou H. Wang L. Figeys D. Rare cell proteomic reactor applied to stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics study of human embryonic stem cell differentiation.Mol. Cell. Proteomics. 2011; 10M110.000679Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 17Maurer M. Müller A.C. Wagner C. Huber M.L. Rudashevskaya E.L. Wagner S.N. Bennett K.L. Combining filter-aided sample preparation and pseudoshotgun technology to profile the proteome of a low number of early passage human melanoma cells.J. Proteome Res. 2013; 12: 1040-1048Crossref PubMed Scopus (29) Google Scholar). However, these methodologies only allow identification of a few hundred proteins at these levels. In this work, we describe a sample preparation approach that utilizes novel small volume focused acoustics-assisted cell lysis, followed by low volume serial reduction, proteolytic digestion and ultra-trace LC-MS analysis. Although two-dimensional separations are often used for deep proteomic analysis, limited sample analysis is best conducted by high peak capacity separation in a single dimension, eliminating potential sample losses from the second dimension. Furthermore, it is known that ultra-low mobile phase flow rates (≤20 nL/min) dramatically improve electrospray signals, as a consequence of improved ionization efficiency (18Valaskovic G.A. Kelleher N.L. McLafferty F.W. Attomole protein characterization by capillary electrophoresis-mass spectrometry.Science. 1996; 273: 1199-1202Crossref PubMed Scopus (343) Google Scholar, 19Zhou F. Lu Y. Ficarro S.B. Adelmant G. Jiang W. Luckey C.J. Marto J.A. Genome-scale proteome quantification by DEEP SEQ mass spectrometry.Nat. Commun. 2013; 4: 2171Crossref PubMed Scopus (83) Google Scholar, 20Luo Q. Tang K. Yang F. Elias A. Shen Y. Moore R.J. Zhao R. Hixson K.K. Rossie S.S. Smith R.D. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry.J. Proteome Res. 2006; 5: 1091-1097Crossref PubMed Scopus (51) Google Scholar, 21Ivanov A.R. Zang L. Karger B.L. Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns.Anal. Chem. 2003; 75: 5306-5316Crossref PubMed Scopus (146) Google Scholar). In prior work, we have shown that reduction of the LC column diameter in a high resolution porous layer open tube (PLOT) [1]The abbreviations used are:PLOTporous layer open tubularCTCcirculating tumor cellDPBSDulbecco's phosphate-buffered salineEMEMEagle's Minimum Essential MediumEPCendothelial progenitor cellHSChematopoietic stem cellKDRkinase insert domain receptor. [1]The abbreviations used are:PLOTporous layer open tubularCTCcirculating tumor cellDPBSDulbecco's phosphate-buffered salineEMEMEagle's Minimum Essential MediumEPCendothelial progenitor cellHSChematopoietic stem cellKDRkinase insert domain receptor. format utilizing ultra-low flow can generate a significant gain in limited sample proteomic profiling capabilities (22Yue G. Luo Q. Zhang J. Wu S.-L. Karger B.L. Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns.Anal. Chem. 2007; 79: 938-946Crossref PubMed Scopus (128) Google Scholar). As shown in the current paper, a combination of PLOT-LC with advanced MS instrumentation and data processing can lead to zeptomole detection sensitivity and quantitation. Furthermore, the integration of all the above steps yields thousands of proteins identified and quantitated from a small number of rare cells (less than one thousand) isolated from 1 ml whole blood. The developed technology opens up the possibility of deep proteomic analysis of rare cells in body fluids. porous layer open tubular circulating tumor cell Dulbecco's phosphate-buffered saline Eagle's Minimum Essential Medium endothelial progenitor cell hematopoietic stem cell kinase insert domain receptor. porous layer open tubular circulating tumor cell Dulbecco's phosphate-buffered saline Eagle's Minimum Essential Medium endothelial progenitor cell hematopoietic stem cell kinase insert domain receptor. All reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO) at the highest purity unless otherwise stated. MCF-7 human breast adenocarcinoma cells (ATCC, Manassas, VA) were cultured in 75 cm2 tissue culture flasks at 37 °C, 5% CO2. MCF-7 cells were incubated in Eagle's Minimum Essential Medium (EMEM; ATCC) supplemented with 10% fetal bovine serum, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 0.01 mg ml−1 bovine insulin. Cells were grown to preconfluence and isolated for experiments by trypsinization using a 0.25% trypsin-EDTA solution. Prior to cell isolation or cell lysis, cells were centrifuged at 200 g for 10 min at 4 °C. The cell culture medium was removed, and MCF-7 cells were washed twice with ice cold 1× Dulbecco's phosphate-buffered saline (DPBS, Sigma-Aldrich) containing 0.5% EDTA (v/v). The cells were resuspended at a concentration of ∼1000 cells/μl, and the cell numbers were counted three times using a hemacytometer and/or flow cytometer (Cell Lab Quanta SC; Beckman Coulter, Brea, CA). Microfluidic channels were fabricated as previously described.(23Plouffe B.D. Njoka D.N. Harris J. Liao J. Horick N.K. Radisic M. Murthy S.K. Peptide-mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow.Langmuir. 2007; 23: 5050-5055Crossref PubMed Scopus (123) Google Scholar, 24Xia Y. Whitesides G.M. Soft lithography.Angew. Chem. Int. Ed. 1998; 37: 550-575Crossref PubMed Google Scholar) Wire arrays were designed using PCB123® printed-circuit board design software and ordered from Sunstone Circuits (Mulino, OR). The wire dimensions were set to provide a gap encompassing the width of the microfluidic channel; the height and width of the wires were set to 35 μm and 178 μm, respectively. Teflon-insulated 18 gauge copper wires were soldered to the ends of each of the printed circuit board arrays, and the arrays were connected to a DC power supply (Elenco Electronics XP-4, Wheeling, IL) that provided three fixed-current settings of 0.25 A, 0.50 A, and 1.00 A via standard alligator clip connectors. The PDMS channels and wire arrays were visually aligned. DynaBeads® MyOneTM Carboxylic Acid magnetic particles (Life Technologies, Carlsbad, CA) were modified with antibodies, either against the epithelial cell adhesion molecule (mouse anti-human EpCAM; Santa Cruz Biotechnology, Santa Cruz, CA) or against CD133 (mouse anti-human CD133, Miltenyi Biotec Inc, Auburn, CA) using standard carbodiimide chemistry (25Hermanson G.T. Bioconjugate Techniques. Academic Press, Boston1996Google Scholar) in ratios suggested by the reagent manufacturer (1:1 molar ratio of beads to protein; Pierce Biotechnology, Rockford, IL). Whole blood was drawn from healthy volunteers and collected in EDTA-coated Vacutainer® tubes (Becton Dickinson, Franklin Lakes, NJ). Approval from the Northeastern University Institutional Review Board was obtained for this purpose (NU IRB #: 11–07-19). The location of the interface between the injected blood and buffer was first evaluated (12Plouffe B.D. Mahalanabis M. Lewis L.H. Klapperich C.M. Murthy S.K. Clinically relevant microfluidic magnetophoretic isolation of rare-cell populations for diagnostic and therapeutic monitoring applications.Anal. Chem. 2012; 84: 1336-1344Crossref PubMed Scopus (81) Google Scholar, 26Plouffe B.D. Lewis L.H. Murthy S.K. Computational design optimization for microfluidic magnetophoresis.Biomicrofluidics. 2011; 5 (013413)PubMed Google Scholar). A Coulter counter/flow cytometer (Cell Lab Quanta™ SC; Beckman Coulter, Brea, CA) was used to count the number of target (MCF-7) cells versus native polymorphonuclear cells that were separated. A protocol based on the distinct size difference of these two cells, was developed to identify each cell population. The cells were gated by their electronic volume and granularity, and the total number of cells within the recovered suspension was determined. Various concentrations of MCF-7 cells (500–100,000 cells) were spiked into 1 ml of whole blood. Following this, 10 μl of modified antiEpCAM magnetic microbeads was added to 1 ml of unprocessed blood and allowed to incubate for 30 min on a rotary mixer. This experiment was conducted at optimized flow rates, as described in the theory section below. For all MCF-7 experiments, the flow rate of the samples was fixed at 240 μl/min, and a center stream of 1× RBC lysis buffer (Ebioscience Inc., San Diego, CA) flowed at 160 μl/ml. Target and nontarget cells were collected in separate methanol cleaned microcentrifuge tubes. To establish an accurate gating of MCF-7 for subsequent cell counts, we first analyzed a homogeneous suspension of approx. 100,000 MCF7 cells. To identify the MCF-7 cells in the flow cytometer, we gated the electronic volume (EV) versus side scattering (SS). We also spiked 100,000 MCF-7 cells into whole blood and ran the sample through the flow cytometer to ensure an accurate gating of the target cells. MCF-7 are distinguishably larger than the surrounding blood cells and thus are the only cells located in the gate. The CV for these calibration samples was 1.2–2.6% (n = 15). On the other hand, gating for the EPCs and HSCs could not be based on cultured homogeneous suspensions. Therefore, buffy coat samples (via Ficoll-Paque density gradient centrifugation) were used to establish standard gates. Gates were first generated with unstained samples (EV and SS and CD34+ only staining to obtain the initial gating from the histograms (CV of 7.5%)). Also, kinase insert domain-containing receptor (KDR)-only (CV = 5.3%, n = 8) and CD45-only (WBC are CD45+; CV = 15.2%, n = 8) were analyzed for their subsequent histograms. These individual gates then allowed for accurate scatter plots based on three-color staining. From these studies, we can assume the CV was between 5.3 and 15.2% for the EPC and HCS cell counting. To illustrate the utility of the magnetophoretic design for isolation of rare cells, we extracted hematopoietic stem cell (HSCs) and endothelial progenitor cells (EPCs) from whole blood using antiCD133 functionalized microparticles. Again, whole blood was drawn from healthy volunteers and collected in EDTA-coated Vacutainer® tubes (Becton Dickinson). Isolated cells were then labeled with antibodies to identify HSC and EPC populations. The HSCs were identified as labeling positive for mouse anti-human CD34 conjugated to fluorescein isothiocyanate (antiCD34-FITC; Santa Cruz) and mouse anti-human CD45 conjugated to phycoerythrin (antiCD45-PE; Santa Cruz), and negative for goat anti-human KDR (kinase insert domain receptor; Santa Cruz). The KDR was then conjugated to a secondary antibody donkey anti-goat peridinin chlorophyll protein (PerCP; R&D Systems, Minneapolis, MN). EPCs were identified as labeling positive for antiCD34-FITC and antiKDR-PerCP, and negative for antiCD45-PE. Both cell populations were distinguished via a flow cytometer (Fig. 6). After separation, the ∼650 μl samples (in RBC lysis buffer) were concentrated using a NdFeB permanent magnet. The samples were rinsed twice with 500 μl of PBS and transferred to a lysis mini-tube. The cell losses because of concentrating, rinsing, and transfer were monitored using at least one replicate isolation to assess starting and ending cell numbers. See supplemental Table S1 for a summary of these results. Other experimental procedures, including LC-MS conditions, are available in the Supplement in the online version of the paper. For the purposes of imaging and verification of cell marker presence, separated cells were concentrated using an NdFeB magnet and resuspended in 100 μl PBS. MCF-7 cells were labeled with mouse antiEpCAM-FITC (Santa Cruz). For the CD133+ cells, we labeled the cells with rabbit antiCD34-FITC, mouse antiCD45-PE, and goat antiKDR, along with a secondary stain with anti-goat Alexa Fluor 350. Again, cells were concentrated using an NdFeB magnet. The cells were then fixed using 2% paraformaldehyde and placed onto a hemacytometer for enumeration of the cells. Approximately 100 cells were evaluated for the number of bead attached to the cells. Sample levels of MCF-7 cells (1000, 2000, 3000, 5000, and 10,000 cells) were spiked into 1 ml of whole human blood and mixed with the Dynal MyOne EpCAM-functionalized magnetic microbeads. As negative controls, human blood, without spiked-in MCF-7 cells, was mixed with the EpCAM-functionalized microbeads. Cells functionalized with magnetic microbeads were isolated from human blood cells using a microfluidic magnitophoretic device, as described (12Plouffe B.D. Mahalanabis M. Lewis L.H. Klapperich C.M. Murthy S.K. Clinically relevant microfluidic magnetophoretic isolation of rare-cell populations for diagnostic and therapeutic monitoring applications.Anal. Chem. 2012; 84: 1336-1344Crossref PubMed Scopus (81) Google Scholar). A flow cytometer was used to count MCF-7 cells. The cells were washed twice with 50 μl of DPBS and processed immediately, as described in the supplement and below. Other experimental procedures and any associated references are available in the Supplement in the online version of the paper. Fig. 1 presents an overview of the platform. For method development, samples consisting of cultured MCF-7 cells spiked into blood at levels of 1000–100,000 MCF-7 cells in 1 ml of whole blood were utilized (Fig. 1A). The magnetophoretic isolation of these cells included incubation of the sample with magnetic beads functionalized with antibodies against EpCAM followed by isolation (12Plouffe B.D. Mahalanabis M. Lewis L.H. Klapperich C.M. Murthy S.K. Clinically relevant microfluidic magnetophoretic isolation of rare-cell populations for diagnostic and therapeutic monitoring applications.Anal. Chem. 2012; 84: 1336-1344Crossref PubMed Scopus (81) Google Scholar). The collected target cells were rinsed and lysed using focused ultrasonication (Covaris), digested with trypsin and analyzed using PLOT nLC coupled to a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) (Fig. 1C). Stable isotope labeled (SIL) peptides were added to the samples after completion of tryptic digestion for targeted quantitative analysis of selected proteins. MS data were processed to enable quantitative proteomic profiling using both label-free and isotope reference-based techniques followed by gene ontology analysis. After development and validation of the platform using MCF-7 cells spiked into whole blood, the optimized workflow was then applied to high specificity isolation from the blood and proteomic characterization of the CD133+ cells from blood (Fig. 1B). A key attribute of this platform is the reduction of the minimum amount of blood necessary to study EPC and HSC and potentially other rare cell populations by at least 1–2 orders of magnitude relative to the current state of the art (27Kim J. Jeon Y.J. Kim H.E. Shin J.M. Chung H.M. Chae J.I. Comparative proteomic analysis of endothelial cells progenitor cells derived from cord blood- and peripheral blood for cell therapy.Biomaterials. 2013; 34: 1669-1685Crossref PubMed Scopus (15) Google Scholar, 28Medina R.J. O'Neill C.L. Sweeney M. Guduric-Fuchs J. Gardiner T.A. Simpson D.A. Stitt A.W. Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities.BMC Med. Genomics. 2010; 3: 18Crossref PubMed Scopus (270) Google Scholar). We first detail the individual steps that lead to the advanced workflow for deep proteomic analysis of rare cells in whole blood. A cornerstone of the platform for deep proteomic profiling of rare cells is the ultra-low flow (ULF) PLOT-nLC column technology. To capture the high sensitivity benefits of ultra-low flow, we used 4 m long 10 μm i.d. poly(styrene-divinylbenzene) (PS-DVB) PLOT columns with ∼1 μm thickness of the permeable layer (Fig. 2A). It has been previously shown that the PLOT columns offer excellent chromatographic efficiencies (22Yue G. Luo Q. Zhang J. Wu S.-L. Karger B.L. Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns.Anal. Chem. 2007; 79: 938-946Crossref PubMed Scopus (128) Google Scholar). The PLOT column was coupled to a monolithic trapping (microSPE) column using a zero dead volume connector. We optimized weight fractions of the PLOT polymerization mixture constituents that resulted in improved column-to-column reproducibility of the PLOT columns and increased hydrophobicity of the monolithic microSPE columns by copolymerizing PS-DVB with 1-decene. Furthermore, the coupling of the PLOT-nLC to an advanced fast duty cycle high resolution mass spectrometer (Q-Exactive) improved the sensitivity and accuracy of quantitation in comparison to our and other previous works (18Valaskovic G.A. Kelleher N.L. McLafferty F.W. Attomole protein characterization by capillary electrophoresis-mass spectrometry.Science. 1996; 273: 1199-1202Crossref PubMed Scopus (343) Google Scholar, 19Zhou F. Lu Y. Ficarro S.B. Adelmant G. Jiang W. Luckey C.J. Marto J.A. Genome-scale proteome quantification by DEEP SEQ mass spectrometry.Nat. Commun. 2013; 4: 2171Crossref PubMed Scopus (83) Google Scholar, 20Luo Q. Tang K. Yang F. Elias A. Shen Y. Moore R.J. Zhao R. Hixson K.K. Rossie S.S. Smith R.D. More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry.J. Proteome Res. 2006; 5: 1091-1097Crossref PubMed Scopus (51) Google Scholar, 21Ivanov A.R. Zang L. Karger B.L. Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns.Anal. Chem. 2003; 75: 5306-5316Crossref PubMed Scopus (146) Google Scholar) (see Supplemental Materials). As a baseline set of experiments to show the potential of the PLOT column-based LC-MS platform for high sensitivity proteomic analysis, the performance of the PLOT column was assessed using a split-injection approach (29Yue G. Luo Q. Zhang J. Wu S.L. Karger B.L. Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns.Anal. Chem. 2007; 79: 938-946Crossref PubMed Scopus (146) Google Scholar). Analyzing an equimolar mixture of digested protein standards (Michrom Bioresources, Auburn, CA, "Bovine 6 Protein Mix," P/N PTD/00001/63, containing beta lactoglobulin, lactoperoxidase, carbonic anhydrase, glutamate dehydrogenase, alpha casein, and serum albumin) with a nontargeted data dependent data acquisition (DDA) method resulted in detection limits down to 10–50 zmol level (S/N>5), based on single stage mass spectrometry (MS1) (Fig. 2B), and unambiguous MS/MS fragment matching (Fig. 2C). A linear MS response was recorded over a dynamic range of over four orders of magnitude, ranging from 10 zmol (S/N>25) to 100
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