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Jessaniniessen2005.Pdf ARTICLES A streamlined platform for high-content functional proteomics of primary human specimens methods Nadim Jessani1,4,5, Sherry Niessen1,5, BinQing Q Wei1,5, Monica Nicolau2, Mark Humphrey1, Youngran Ji2, Wonshik Han3, Dong-Young Noh3, John R Yates III1, Stefanie S Jeffrey2 & Benjamin F Cravatt1 .com/nature e Achieving information content of satisfactory breadth and depth resolution, facilitating the identification of low-abundance proteins remains a formidable challenge for proteomics. This problem is in complex samples8,9. These techniques, however, are time- .natur w particularly relevant to the study of primary human specimens, consuming (many hours per sample), difficult to perform in such as tumor biopsies, which are heterogeneous and of finite parallel and require large quantities of proteome (B1mgor quantity. Here we present a functional proteomics strategy that greater) for optimal analysis. Alternative proteomic approaches unites the activity-based protein profiling and multidimensional http://ww have emerged that have greater throughput (matrix-assisted laser protein identification technologies (ABPP-MudPIT) for the desorption ionization (MALDI) imaging6 and surface-enhanced 7 oup streamlined analysis of human samples. This convergent laser desorption ionization time-of-flight (SELDI-TOF) spectro- r G platform involves a rapid initial phase, in which enzyme activity metry), albeit at the expense of sensitivity. Furthermore, each of signatures are generated for functional classification of samples, these methods, by focusing on measurements of protein expression, followed by in-depth analysis of representative members from provides only an indirect readout of protein activity, which is lishing each class. Using this two-tiered approach, we identified more regulated by a myriad of post-translational events in vivo10. b than 50 enzyme activities in human breast tumors, nearly a The troublesome trade-off between the breadth and depth of Pu third of which represent previously uncharacterized proteins. information procurable by different techniques has frustrated Comparison with cDNA microarrays revealed enzymes whose efforts to implement an efficient, systematic strategy for high- Nature activity, but not mRNA expression, depicted tumor class, content proteomics. These concerns are particularly relevant to the 5 underscoring the power of ABPP-MudPIT for the discovery of new study of primary human specimens, which are often heterogeneous 11 200 markers of human disease that may evade detection by other and of limited quantity , and therefore mandate interrogation by © molecular profiling methods. proteomic methods that can achieve both high throughput (to deal with sample heterogeneity) and sensitivity (to identify rarely Postgenomic research promises to deliver humankind into the age expressed and post-translationally regulated proteins in samples of molecular medicine, where diseases are diagnosed and treated of finite amount). through the detection and pharmacological targeting of pathology- Toaddress these important issues, we have introduced a chemical linked proteins1. The success of this biomedical vision hinges on the technology, referred to as activity-based protein profiling rapid translation of genomic information into the discovery of new (ABPP)12,13 that uses active site–directed probes to read out the protein biomarkers and therapeutic targets. Proteomics aims to functional state of many enzymes directly in whole proteomes. facilitate this process by developing new methods for the parallel ABPP probes selectively label active enzymes, but not their inactive analysis of many proteins in samples of high biological complexity2. (for example, zymogen or inhibitor-bound) forms, facilitating the To date, several strategies have emerged for the large-scale characterization of changes in enzyme activity that occur without discovery of differentially expressed proteins in cells, tissues and corresponding alterations in protein or transcript expression14,15. fluids, including two-dimensional gel electrophoresis (2DE)3, Additionally, because ABPP probes label enzymes based on shared liquid chromatography–mass spectrometry (LC-MS; for example, catalytic properties rather than mere expression level, they provide isotope-coded affinity tagging (ICAT)4 and multidimensional- exceptional access to low-abundance portions of the proteome, protein identification technology (MudPIT)5) and MS-based pro- which can be read out even in simple formats like one-dimensional teomic pattern analysis6,7. A survey of the data acquired using each gel electrophoresis (1DE)14. Using such 1DE-ABPP methods, hun- of these approaches reveals an intriguing paradox. Multidimen- dreds of proteomes can be analyzed per day by a single academic sional separation methods have remarkable sensitivity and group. These features have allowed the routine application 1The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037, USA. 2Department of Surgery, Stanford University School of Medicine, MSLS Building, Room P214, 1201 Welch Road, Stanford, California 94305, USA. 3Cancer Research Institute and Department of Surgery, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, South Korea. 4Present address: Celera, 180 Kimball Way, South San Francisco, California 94080, USA. 5These authors contributed equally to this work. Correspondence should be addressed to B.F.C. ([email protected]). PUBLISHED ONLINE 23 AUGUST 2005; DOI:10.1038/NMETH778 NATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 691 ARTICLES BC100BC103BC104BC106BC108BC110BC112BC114 Rh Phase I 1DE-ABPP: Rh generation of Hierarchical enzyme activity Rh clustering signatures BC100 BC101 BC102 BC103 BC104 BC105 BC106 BC107 In-gel fluorescence BC108 BC109 BC110 BC111 BC112 BC113 BC114 BC115 BC116 BC117 BC118 BC119 BC120 Probe-labeled analysis proteome N C C B N methods m/z N C N C N N B Phase II C C C N ABPP-MudPIT: N On-bead B Avidin B Target m/z LC-MS/MS N B in-depth analysis identification N digest affinity C C and C purification .com/nature quantification e B m/z Tryptic peptides Probe-labeled proteome .natur w Figure 1 | Integration of the ABPP and MudPIT methods for high-content functional proteomics of primary human specimens. A two-tiered platform is presented. In the first phase, proteomic samples are treated with a rhodamine (Rh)-tagged ABPP probe and resolved by 1DE to provide enzyme activity signatures (visualized by in-gel fluorescence scanning). Hierarchical clustering of these molecular profiles is then used for the functional classification of http://ww samples. In the second phase, representative members of each sample class are treated with a biotinylated ABPP probe (B) and the labeled proteins are enriched by binding to avidin-conjugated beads and subjected to on-bead trypsin digestion. The resulting tryptic peptide mixtures are analyzed by multidimensional 16,17 oup LC-MS (MudPIT), and the levels of specific enzyme activities are estimated by spectral counting . r G of ABPP to many biological samples in parallel, permitting, for spectral counting of probe-enriched enzymes might address this lishing b example, the classification of human cancer cell lines into pheno- final issue. We set out to test this integrated ABPP-MudPIT plat- Pu typically relevant groups based on their shared enzyme activity form by comparatively profiling a set of 33 primary human breast profiles14. Nonetheless, despite these valuable attributes, the inher- tumor and normal breast tissue biopsies. Clinical parameters such ent resolution and sensitivity limits of 1DE have, to date, precluded as estrogen receptor (ER) and progesterone receptor (PR) status Nature comprehensive access to many of the enzyme activities present in were determined for tumor samples to assist in the interpretation of 5 individual proteomic samples. proteomic profiles (see Supplementary Table 1 online for a list of 200 Here we present an advanced platform that unites the ABPP and clinical data). © MudPIT technologies, providing a general and efficient route for achieving both breadth and depth in functional proteome analysis. Phase I: rapid analysis of breast tumors by 1DE-ABPP We apply this integrated approach to more than 30 primary human For tumor profiling experiments, we used fluorophosphonate breast tumor and normal breast specimens, and find several (FP)-based ABPP probes that target the serine hydrolase super- enzyme activities that are elevated in specific breast tumor classes. family14,18 (see Supplementary Fig. 1 online for structures of FP probes). Serine hydrolases are a large and diverse class of enzymes RESULTS that comprise about 1% of the human proteome, including several A tiered platform for high-content functional proteomics enzymes implicated in cancer19–21. For the efficient analysis of large numbers of primary human tissue We carried out the phase I analysis using 5–10-mm frozen tissue specimens, we envisaged a two-tiered platform (Fig. 1). In a rapid sections, portions of which were also allocated for cDNA micro- initial phase, 1DE-ABPP would be used to generate enzyme activity array experiments to allow a direct comparison of transcript and signatures for the functional classification of proteomic samples enzyme activity profiles. Homogenized tissue sections (soluble and (phase I). ABPP-MudPIT would then be performed on representa- membrane; 12 mg protein/sample) were treated with a rhodamine-
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