Sample multiplexing for targeted pathway proteomics in aging mice Qing Yua, Haopeng Xiaoa,b, Mark P. Jedrychowskia,b, Devin K. Schweppea, Jose Navarrete-Pereaa, Jeffrey Knottc, John Rogersd, Edward T. Chouchania,b, and Steven P. Gygia,1 aDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; bDepartment of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115; cCell Signaling Technologies, Danvers, MA 01923; and dThermo Fisher Scientific, Rockford, IL 61101 Edited by Anne Brunet, Stanford University, Stanford, CA, and accepted by Editorial Board Member K. C. Garcia March 18, 2020 (received for review November 5, 2019) Pathway proteomics strategies measure protein expression changes High-throughput and precise quantitation of protein pathways in specific cellular processes that carry out related functions. Using is essential for systematic understanding the molecular basis for targeted tandem mass tags-based sample multiplexing, hundreds of complex physiological adaptation. This is perhaps best exempli- proteins can be quantified across 10 or more samples simultaneously. fied in the case of chronic physiological changes that occur with To facilitate these highly complex experiments, we introduce a strat- age. Tissue aging is characterized by a homeostatic decline in a egy that provides complete control over targeted sample multiplex- variety of physiological and psychological functions and is a ing experiments, termed Tomahto, and present its implementation major risk factor in many diseases. There has been a continuous on the Orbitrap Tribrid mass spectrometer platform. Importantly, this effort to understand the underlying cellular and molecular causes, and software monitors via the external desktop computer to the data various hypotheses exist (12). Changes in the genome, epige- stream and inserts optimized MS2 and MS3 scans in real time based nome, and transcriptome have all been observed and, as a result, on an application programming interface with the mass spectrome- genome instability, gene methylation levels, and transcriptional ter. Hundreds of proteins of interest from diverse biological samples changes have all been implicated in aging (13–15). In addition, can be targeted and accurately quantified in a sensitive and high- metabolism remodeling, inflammatory response, and the homeo- throughput fashion. It achieves sensitivity comparable to, if not bet- stasis of multiple organelles were frequently implicated (16, 17). ter than, deep fractionation and requires minimal total sample input However, little work has been done to investigate proteome changes (∼10 μg). As a proof-of-principle experiment, we selected four path- (18, 19) and, surprisingly, only a minimal disturbance was reported in CHEMISTRY ways important in metabolism- and inflammation-related processes the tissues studied (20). In addition to a lack of proteomic charac- (260 proteins/520 peptides) and measured their abundance across 90 terization in aging organisms, current knowledge suggests different samples (nine tissues from five old and five young mice) to explore tissues in an organism share common altered pathways due to aging effects of aging. Tissue-specific aging is presented here and we high- but also have their own unique responses which necessitate tissue- light the role of inflammation- and metabolism-related processes in specific investigation (21, 22). For example, Christensen et al. found white adipose tissue. We validated our approach through compari- strikingly different gene methylation profiles in multiple human tissues son with a global proteome survey across the tissues, work that we SYSTEMS BIOLOGY also provide as a general resource for the community. Significance targeted pathway proteomics | tissue-specific aging | isobaric labeling | A user-friendly workflow, termed Tomahto, enables real-time real-time instrument control | Tomahto targeted pathway proteomics assays using two-dimensional multiplexing technology. Hundreds of proteins of interest n proteomics, both targeted and untargeted approaches are from a multitude of samples can be targeted and accurately Iemployed depending on the experiment’s goal (1). While the quantified in a remarkably sensitive fashion. We highlight untargeted approach is widely used in a discovery phase to Tomahto’s ease of use, sensitivity, and accuracy and present identify potential protein signatures that underlie cellular pro- proof-of-principle utility by targeting 260 metabolism- and cesses (2), it often requires substantially more starting material, inflammation-related proteins across 90 samples (nine tissues extensive sample fractionation, and more instrument time rela- from five old and five young mice). Tissue-specific aging effects tive to targeted approaches (3, 4). It also can suffer from lower are presented with specific interest in metabolic and in- sampling reproducibility due to its stochastic nature. In contrast, flammatory pathways of white adipose tissue. We validated targeted approaches are favored if a preselected set of proteins our approach through comparison with a global proteome needs to be consistently measured across a large cohort of samples, survey across the tissues, work that we also provide as a such as in clinical settings, or when sample amount is limited (5). To general resource for the community. further improve throughput and reduce quantitative variability, two-dimensional (2D) multiplexing (i.e., multiplexing peptide tar- Author contributions: Q.Y., M.P.J., E.T.C., and S.P.G. designed research; Q.Y., H.X., J.N.-P., and J.K. performed research; Q.Y., D.K.S., and J.R. contributed new reagents/analytic gets and multiplexing samples) can be achieved via isobaric labeling tools; Q.Y. analyzed data; and Q.Y. and S.P.G. wrote the paper. strategies, such as using tandem mass tags (TMT) (6). Although the The authors declare no competing interest. quantitative accuracy using isobaric tags can be distorted by This article is a PNAS Direct Submission. A.B. is a guest editor invited by the cofragmenting peptide species within the isolation window (7, 8), it Editorial Board. can be readily restored by using synchronous precursor selection This open access article is distributed under Creative Commons Attribution-NonCommercial- and MS3 scans (SPS-MS3) (9). Previously, our laboratory in- NoDerivatives License 4.0 (CC BY-NC-ND). troduced a 2D multiplexing method (triggered by offset, multi- Data deposition: All MS raw files haven been deposited to the PRIDE archive with the plexed, accurate-mass, high-resolution, and absolute quantification identifier PXD017385. —TOMAHAQ), which incorporates TMT and SPS-MS3 with See online for related content such as Commentaries. targeted proteomics. The method exploits the use of synthetic 1To whom correspondence may be addressed. Email: [email protected]. trigger peptides, which completely obviates the need for peptide This article contains supporting information online at https://www.pnas.org/lookup/suppl/ detection prior to quantification. However, the method requires a doi:10.1073/pnas.1919410117/-/DCSupplemental. very cumbersome method structure, limiting its application (10, 11). First published April 24, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1919410117 PNAS | May 5, 2020 | vol. 117 | no. 18 | 9723–9732 Downloaded by guest on September 29, 2021 (21) and Hudgins et al. reported highly variable expression of peptides to measure kinase levels across these lines (Fig. 2A). A senescence biomarkers in age- and tissue-specific manners (23). TMT10plex was created consisting of biological triplicates of In this paper we present an application programming interface HCT116 and MCF7 and quadruplicates of HEK293T cells. (API)-based algorithm, termed Tomahto, which enables real- Synthetic peptides were readily available for 77 kinases (154 time instrument control and decision making. Tomahto pro- peptides; see Dataset S1) that could be used in a Tomahto assay vides an array of functionalities including MS1 peak detection, to quantify kinase levels across these three cell lines. The sen- MS2 real-time peak matching (RTPM), MS2 fragmentation sitivity of Tomahto was exemplified by peptide FADIVSILDK pattern match, SPS ion purity filter, MS3 automatic gain control, from EPHA1 (Fig. 2B). The trigger peptide was readily detected MS3 quant scan insertion, and target peptide closeout. Re- and its sequence confirmed in real time by peak matching, markably, the only required input from a user is a list of peptide whereas the precursor of its endogenous counterpart was un- targets, greatly simplifying the experimental setup. We first detectable in MS1 scans. However, Tomahto allowed extensive benchmarked the technology with three well-established human yet specific accumulation of signals (>800 ms) to confirm its cell lines by targeting 154 peptides corresponding to 77 kinases. presence by an Orbitrap MS2 spectrum which resulted in a Next, to enable targeted protein profiling of age-related meta- subsequent successful quantification event. Seventy-one of the 77 bolic and inflammatory changes, we created a large Tomahto targeted kinases were detected and quantified from a 2-h gra- assay (520 peptides corresponding to 260 proteins). The assay can dient of the unfractionated mixture (2 h total) (Fig. 2C). The be readily adopted by any TMT-based targeted analysis in murine average coefficient of variation for replicates from the three cell systems. We demonstrate that white adipose tissue (WAT) was by lines was 3.7 ± 2.9%