Large-Scale Filter-Aided Sample Preparation Method for the Analysis

Mar 5, 2017 - during IAP, here we developed a large-scale FASP method (LFASP) for ... ubiquitinome analysis, LFASP overcomes the general limitation in...
0 downloads 0 Views 1011KB Size
Subscriber access provided by University of Newcastle, Australia

Technical Note

A large-scale filter-aided sample preparation method (LFASP) for the analysis of the ubiquitinome Albert Casanovas, Roberto Pinto-Llorente, Montserrat Carrascal, and Joaquín Abian Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04804 • Publication Date (Web): 05 Mar 2017 Downloaded from http://pubs.acs.org on March 6, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A large-scale filter-aided sample preparation method (LFASP) for the analysis of the ubiquitinome Albert Casanovas1, Roberto Pinto-Llorente1, Montserrat Carrascal1 and Joaquín Abián1,2,* 1

Proteomics Laboratory CSIC/UAB, Institute of Biomedical Research of Barcelona, Spanish National

Research Council (IIBB-CSIC/IDIBAPS), E-08036 Barcelona, Spain. 2

Autonomous University of Barcelona, E-08193 Bellaterra, Spain.

*Contact information for corresponding author: Joaquín Abián Proteomics Laboratory CISC/UAB Institute of Biomedical Research of Barcelona. Spanish National Research Council Rosselló 161, 6ª planta 08036 Barcelona Spain Tel.: (+34) 93 581 48 53 Fax: (+34) 93 581 49 13 E-mail: [email protected]

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Protein ubiquitination regulates key cellular functions, including protein homeostasis and signal transduction. The digestion of ubiquitinated proteins with trypsin yields a glycine-glycine remnant bound to the modified lysine residue (K-ε-GG) that can be recognized by specific antibodies for immunoaffinity purification (IAP) and subsequent identification of ubiquitination sites by mass spectrometry. Previous ubiquitinome studies based on this strategy have consistently digested milligram amounts of protein as starting material using in-solution digestion protocols prior to K-ε-GG enrichment. Filter-aided sample preparation (FASP) surpasses in-solution protein digestion in cleavage efficiency, but its performance has thus far been shown for digestion of sample amounts on the order of micrograms. Because cleavage efficiency is pivotal in the generation of the K-ε-GG epitope recognized during IAP, here we developed a large-scale FASP method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASP-based tryptic digestion is efficient, robust, reproducible and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and show an ~3-fold reduction in the proportion of miscleaved peptides with the method presented here. Beyond ubiquitinome analysis, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a variety of applications that demand a large(r) amount of starting material.

ACS Paragon Plus Environment

Page 2 of 17

Page 3 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Introduction Protein ubiquitination is an essential post-translational modification that regulates key cellular functions, such as protein degradation through the ubiquitin-proteasome system, signal transduction, and DNA replication and repair1,2. Ubiquitination involves the covalent attachment of the 76-amino acid ubiquitin protein through its C-terminus to the ε-amino group of a lysine residue in the substrate protein. Large-scale detection of ubiquitination sites by mass spectrometry requires specific enrichment of peptides containing the modified lysine of ubiquitinated proteins. A strategy for this enrichment extensively used in contemporary studies of the ubiquitin-modified proteome (i.e., ubiquitinome) employs a specific antibody3,4 for immunoaffinity purification (IAP) of peptides containing the glycine-glycine (GG) remnant (derived from the ubiquitin Cterminus) that remains on the ubiquitinated lysine residue (K-ε-GG) after proteolytic digestion with trypsin. The GG remnant prevents cleavage by trypsin5,6 resulting in an internal modified lysine residue in a previously ubiquitinated peptide and therefore serves as a signature of ubiquitination that allows identification of the specific modification site using mass spectrometry. This strategy has been successfully used in largescale studies to determine the dynamics of the cellular ubiquitinome in a variety of conditions including infection7, oncogene-induced senescence8, Hsp90 inhibition9, and proteasome inhibition3,10. Importantly, efficient proteolytic digestion is the cornerstone of this approach since the K-ε-GG epitope recognized by the antibody is generated during tryptic digestion of the proteome. Ubiquitinome studies have thus far used large amounts of starting material and urea-based in-solution digestion for sample preparation3,7-12. An alternative to in-solution digestion is the use of ultrafiltration units as “proteomic reactors” for chemical modification and digestion of proteins. This approach, initially introduced by Manza et al.13, was further developed and termed filter-aided sample preparation (FASP) by Wisniewsky and co-workers14. FASP has been extensively used in a variety of sample types and applications15-17 and has been the subject of recent developments and evaluation18,19. FASP allows the use of large amounts of SDS to achieve virtually complete proteome solubilization followed by efficient removal of the detergent through sequential washes with urea in the ultrafiltration unit14. In addition to the advantages in protein solubilization, recent studies have shown that FASP surpasses in-solution digestion in cleavage efficiency when trypsin is used as the proteolytic enzyme20,21. These results suggest FASP as an attractive alternative to in-solution digestion for ubiquitinome studies using the K-ε-GG enrichment strategy. The performance of FASP-based methods has, however, thus far been shown for the digestion of samples on the order of micrograms, whereas ubiquitinome studies consistently use milligram amounts of protein as starting material3,7-12. To overcome this discrepancy, we devised a large-scale FASP-based method (LFASP) for digestion of milligram amounts of protein and evaluated its applicability to the study of the ubiquitinome. Our results demonstrate that LFASPbased tryptic digestion is efficient, robust, reproducible and applicable to the study of the ubiquitinome. We benchmark our results with state-of-the-art ubiquitinome studies and demonstrate the superior cleavage efficiency of the method presented here.

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Experimental section Cell culture Jurkat T-lymphocytes (clone E6-1) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, St Louis, MO, USA), supplemented with 10 % (v/v) complement-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA). For ubiquitinome analysis in the context of proteasome inhibition, cells were treated with 1 µM Bortezomib (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in RPMI 1640 medium supplemented with 2 % (v/v) complement-inactivated FBS for 4 h at a density of 2 x 106 cells/mL. After treatment, cells were washed twice with PBS and cell pellets were frozen and stored at -80 °C. Cell lysis Cells were resuspended in lysis buffer (25 mM TEAB pH 8.5, 4 % SDS) and lysed by pulse sonication. Following lysis, an aliquot was withdrawn for determination of protein concentration using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) and the remaining sample was incubated 5 min at 95 °C after addition of DTT (100 mM, final concentration). Lysates were subsequently clarified by centrifugation. Filter-aided sample preparation Proteins were digested in 15 mL ultrafiltration units (Amicon Ultra 15, 10 kDa NMWL, Merck Millipore, Billerice, MA, USA). All centrifugations were carried out at 13 °C and 4,000 x g. Prior to use, the ultrafiltration units were conditioned by addition of 2.4 mL of 100 mM NaOH and centrifugation (10 min), followed by a wash with 2.4 mL of water and centrifugation (15 min). The filter units were subsequently passivated by overnight incubation in 5 % (v/v) Tween-20 as described18 to reduce sample losses. The filter units were then rinsed thoroughly with water and, immediately before sample loading, washed with 12 mL of water and centrifuged for 15 min. Ten aliquots of cell lysate (1.2 mg each) were prepared and digested in parallel in separate filter units. The sample amount that can be loaded in the 15 mL ultrafiltration unit (1.2 mg) was estimated following the ratio sample amount to filter surface commonly used with regular (i.e., small) filters in standard FASP protocols. To remove SDS from the lysates, samples were diluted 1/40 in UT buffer (8 M urea, 100 mM Tris-HCl pH 8.5) and loaded in the passivated filters in two steps, centrifuged for 30 min and the flow-through was discarded. Then, 2 mL of UT buffer was added to the filter unit followed by centrifugation (15 min). Proteins were alkylated by addition of 2 mL of 50 mM iodoacetamide in UT buffer and incubation for 20 min in the dark. Following alkylation, the filter units were centrifuged for 20 min and washed thrice with UT buffer. To remove urea, three buffer exchange steps were performed by addition of 2 mL of 20 mM NH4HCO3 followed by centrifugation (20 min). Then, proteins were digested with trypsin (1:20 w/w, unless otherwise indicated) (Promega, Madison, WI, USA) in 2 mL of 20 mM NH4HCO3 for 14 h at 37 °C. Peptides were collected in a new collection tube by centrifugation (30 min) followed by two washes with 2 mL of 20 mM NH4HCO3. Digests were acidified with TFA (1 % v/v, final concentration). An aliquot from each digest was taken for single-shot proteomic analysis, and the remaining volume of each digest was pooled and lyophilized.

ACS Paragon Plus Environment

Page 4 of 17

Page 5 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For comparison, standard (normal-scale) FASP digestion was carried out in small filters (Amicon Ultra 0.5, 10 kDa NMWL, Merck Millipore, Billerice, MA, USA) following the same procedure. Peptide fractionation by basic pH reversed-phase (bRP) liquid chromatography Peptides were off-line fractionated by bRP essentially as described22 using a Waters XBridge BEH C18 5 µm 4.6 × 250 mm column (Waters, Milford, MA, USA) on an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) operating at 1 mL/min. Briefly, peptides were resuspended in 3 mL of bRP buffer A (5 mM ammonium formate pH 10, 2 % (v/v) ACN), separated into 3 x 1 mL aliquots and fractionated in 3 successive injections. Peptides were eluted by a non-linear gradient of bRP buffer B (5 mM ammonium formate pH 10, 90 % (v/v) ACN). Fractions were collected at 2 min intervals. Twenty-five fractions collected after the flow-through were pooled in a non-contiguous manner to generate five final fractions for K-ε-GG enrichment (Figure 1). An aliquot of the original twenty-five bRP fractions was withdrawn for comprehensive proteomic analysis. Fractions were dried in a SpeedVac concentrator. Anti-K-ε-GG antibody cross-linking Peptides containing the K-ε-GG motif were enriched using the anti-K-ε-GG antibody from the PTMScan ubiquitin remnant motif (K-ε-GG) kit (Cell Signaling Technology, Danvers, MA, USA). The antibody is provided noncovalently bound to protein A agarose beads. To minimize the interference that antibody-derived contaminants have on the enrichment and detection of K-ε-GG peptides10, the antibody was cross-linked to the beads as described elsewhere22. In short, the anti-K-ε-GG antibody beads were washed three times with 1 mL of 100 mM sodium borate pH 9. Following the washes, the antibody beads were resuspended in 1 mL of 20 mM dimethyl pimelimidate and incubated with rotation for 30 min at room temperature to complete the crosslinking step. The antibody beads were subsequently washed twice with 1 mL of 200 mM ethanolamine pH 8 and blocked by incubation in 1 mL of 200 mM ethanolamine pH 8 with rotation for 2 h at 4 °C. After blocking, the antibody beads were washed three times with 1.5 mL of IAP buffer (50 mM MOPS pH 7.2, 10 mM Na2HPO4, 50 mM NaCl), then resuspended in IAP buffer and stored at 4°C. IAP of K-ε-GG peptides K-ε-GG peptides were enriched in the five final bRP fractions using the cross-linked antibody beads as previously described22, with minor modifications. Briefly, peptides from each bRP fraction were dissolved in 1.5 mL of IAP buffer, centrifuged (15,000 x g, 5 min, 4 °C) and then the supernatant was incubated (2 h at 4 °C with rotation) with an amount of cross-linked antibody equivalent to 1/8 of the commercial vial, as recommended10. Following incubation, the antibody beads were washed twice with IAP buffer and three times with 20 mM NH4HCO3. Bound peptides were eluted with 2 x 50 µL of 0.15 % (v/v) TFA. LC-MS/MS analysis Peptides were analyzed by LC-MS/MS using an HPLC system composed of an Agilent 1200 capillary nano pump, a binary pump, a thermostated microinjector and a microswitch valve coupled to an LTQ Orbitrap XL mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray source (Proxeon, Odense, Denmark, now Thermo Scientific). Two micrograms of peptides (single-shot and comprehensive proteomic analyses) or 30 % of the IAP eluate (analysis of K-ε-GG peptides) were dried and resuspended in 20 µL of 1 % (v/v) formic acid, 5 % (v/v) methanol. Peptides were separated with a C18 pre-concentration cartridge (Agilent

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Technologies, Santa Clara, CA, USA) connected to a C18 100 µm x 150 mm column (Nikkyo Technos Co, Tokyo, Japan) at 400 nL/min using a linear gradient (120 min in the comprehensive proteomic analysis -25 bRP fractions- and 360 min in other analyses) from 0 to 35 % solvent B (Solvent A: 0.1 % (v/v) formic acid; solvent B: ACN 0.1 % (v/v) formic acid). The source was operated in positive ion mode at 1.5 kV and with the capillary at 200 °C. The LTQ Orbitrap XL was operated in data-dependent mode at a target mass resolution of 60,000 (at m/z 400). The scan range of each survey scan was m/z 400-1800, and up to the 10 most intense peaks with charge ≥2 and an intensity above the 5,000 threshold were selected and fragmented by collision induced dissociation with normalized collision energy of 35. The maximum injection times for the survey scan and the MS/MS scan were 1 s and 200 ms, respectively, and the respective ion target values were set to 2E5 and 1E4. To minimize the redundant selection of precursor ions, the fragmented ions were dynamically excluded for 45 s (comprehensive proteomic analysis) or 60 s (other analyses). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE23 partner repository with the dataset identifier PXD005421. Data analysis The raw data were processed using Proteome Discoverer (version 1.4.0.288, Thermo Scientific). The fragmentation spectra were searched with the SEQUEST engine against the UniProtKB/Swiss-Prot human database (canonical and isoform sequences, release 2016_05) using the following parameters: trypsin, maximum 3 missed cleavages, 20 ppm precursor mass tolerance, 0.6 Da fragment mass tolerance, cysteine carbamidomethylation as a fixed modification, and oxidation of methionine, GG addition to lysine and acetylation of N-terminal as dynamic modifications. Peptide spectral matches were filtered at 1 % false discovery rate using Percolator24,25. The localization probability for each K-ε-GG site was calculated using ptmRS26 and only K-ε-GG peptides with all sites localized with a probability ≥75 % were kept for further analyses. Peptides with a K-ε-GG site mapped to a peptide C-terminal lysine residue were removed from the dataset except when the site coincided with the protein C-terminal residue. Peptide abundance was estimated by extracting the areas of the precursor ions with the Proteome Discoverer software. To accurately evaluate the quantitative reproducibility, the raw files were analyzed using Progenesis QI for proteomics (v3.0, Nonlinear Dynamics). Briefly, the LC-MS/MS maps of runs within each experiment were aligned. Four (out of 30) runs were excluded from the analysis due to poor alignment (alignment score 90 % of non-K-ε-GG peptides and peptides with one K-ε-GG site are fully cleaved (0 and ≤1 missed cleavages, respectively) in our LFASP dataset, whereas the percentage of miscleaved peptides is at least ~3-fold higher in the reference datasets (Figure 3A and Table S2). The same conclusions can be drawn from data derived from peptide abundance (Figure 3B and Table S2). We note that fully cleaved K-ε-GG peptides have as many missed cleavages as K-ε-GG sites (except for K-ε-GG sites located (i) in the C-terminus of the protein or (ii) at the N-terminal side of a proline residue, which is not considered as a missed cleavage). Overall, these results indicate the superior cleavage efficiency of LFASP over urea-based in-solution digestion protocols used in state-of-the-art ubiquitinome studies. We note that the in-solution digestion protocols in the studies used for comparison employed a lower trypsin-to-protein ratio than that used in LFASP. We evaluated the digestion efficiency of LFASP at different trypsin-to-protein ratios and observed a minor contribution of this factor to the optimized digestion efficiency of LFASP (Figure S2). On the other hand, in this study, we identify more peptides in our K-ε-GG enriched sample (16,256 peptides) than in the dataset of Quadroni et al. (7,978 peptides) and less than in the dataset of Udeshi et al. (75,432 peptides), likely due to differences in the instrumentation used and the extent of peptide fractionation as well as the number of samples and the additional condition (treatment with the deubiquitinase inhibitor PR-619) analyzed by Udeshi et al.10. Since the occurrence of missed cleavages can be stochastic28, miscleaved peptides can be expected to be low in abundance and therefore be more represented in more in-depth proteomic studies. To further investigate if the higher percentage of peptides with missed cleavages in the dataset of Udeshi et al. was due to the digestion method or the depth of the proteomic analysis, we carried out a comprehensive analysis of the proteome by analyzing the 25 fractions collected during bRP fractionation (Figure 1). This analysis quantified 61,031 unique peptides with a dynamic range that matched that of the dataset of Udeshi et al. (Figure S3) and therefore could be used for comparison of cleavage efficiencies. Notably, the distribution of missed cleavages as a function of peptide abundance in the digest shows that the most abundant peptides exhibit a higher percentage of fully cleaved peptides whereas for low-abundance peptides the percentage of peptides with missed cleavages is higher (Figure 4). These data confirm that comprehensive studies are more likely to identify a higher percentage of miscleaved peptides. Importantly, the average percentage of fully cleaved peptides in this dataset is 87 %, and even the percentage of fully cleaved peptides (76 %) in the bin of least abundant peptides (log10 area of precursor ion ≤5.5) is higher than that of non-K-ε-GG peptides in the dataset of Udeshi et al. (71 %) (Figure 3A and Table S2). These results corroborate the higher digestion efficiency of the LFASP method presented here and therefore support the convenience of this approach for protein digestion in ubiquitinome studies.

ACS Paragon Plus Environment

Page 8 of 17

Page 9 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Conclusions Efficient digestion of proteins into peptides is desirable for any proteomic application as it can improve quantification accuracy, sensitivity and reproducibility27, but it is of utmost importance for certain applications, such as the study of the ubiquitinome using IAP of K-ε-GG peptides, because the epitope recognized by the antibody is generated during tryptic digestion of the proteome. Although recent studies have shown that FASP surpasses the cleavage efficiency of in-solution digestion protocols, thus far, ubiquitinome studies have used in-solution digestion protocols likely due to the large amounts of starting material used and the limited sample capacity of standard FASP protocols. Here, we introduced a robust, reproducible and efficient LFASP method and show its applicability to the study of the ubiquitinome. We demonstrate the superior cleavage efficiency of this method over in-solution digestion used in state-of-the-art ubiquitinome studies. Notably, LFASP overcomes the general limitation in sample capacity of standard FASP-based protocols and can therefore be used for a range of applications that require milligram amounts of starting material, including the study of protein acetylation (not shown) or other substoichiometric PTMs in large scale.

Associated content Supporting information includes three figures and two tables.

Acknowledgments We thank Vanessa Casas and Óscar Gallardo for technical assistance. This work was supported by grant BIO2013-46492-R from the Spanish Ministry of Economy and Competitiveness. AC was the recipient of a Beatriu de Pinós Fellowship (2014 BP-B 00168) supported by the Universities and Research Secretariat of the Department of Economy and Knowledge of the Government of Catalonia and by the FP7 of the European Union through the Marie Curie Actions COFUND Programme. The Proteomics Laboratory CSIC/UAB is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013-2016, funded by ISCIII and FEDER.

References (1) Chen, Z. J.; Sun, L. J. Mol Cell 2009, 33, 275-286. (2) Grabbe, C.; Husnjak, K.; Dikic, I. Nat Rev Mol Cell Biol 2011, 12, 295-307. (3) Kim, W.; Bennett, E. J.; Huttlin, E. L.; Guo, A.; Li, J.; Possemato, A.; Sowa, M. E.; Rad, R.; Rush, J.; Comb, M. J.; Harper, J. W.; Gygi, S. P. Mol Cell 2011, 44, 325-340. (4) Xu, G.; Paige, J. S.; Jaffrey, S. R. Nat Biotechnol 2010, 28, 868-873. (5) Seyfried, N. T.; Xu, P.; Duong, D. M.; Cheng, D.; Hanfelt, J.; Peng, J. Anal Chem 2008, 80, 4161-4169. (6) Shi, Y.; Xu, P.; Qin, J. Mol Cell Proteomics 2011, 10, R110 006882. (7) Fiskin, E.; Bionda, T.; Dikic, I.; Behrends, C. Mol Cell 2016, 62, 967-981. (8) Bengsch, F.; Tu, Z.; Tang, H. Y.; Zhu, H.; Speicher, D. W.; Zhang, R. Cell Cycle 2015, 14, 1540-1547. (9) Quadroni, M.; Potts, A.; Waridel, P. J Proteomics 2015, 120, 215-229. (10) Udeshi, N. D.; Svinkina, T.; Mertins, P.; Kuhn, E.; Mani, D. R.; Qiao, J. W.; Carr, S. A. Mol Cell Proteomics 2013, 12, 825-831.

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(11) Wagner, S. A.; Beli, P.; Weinert, B. T.; Nielsen, M. L.; Cox, J.; Mann, M.; Choudhary, C. Mol Cell Proteomics 2011, 10, M111 013284. (12) Wagner, S. A.; Beli, P.; Weinert, B. T.; Scholz, C.; Kelstrup, C. D.; Young, C.; Nielsen, M. L.; Olsen, J. V.; Brakebusch, C.; Choudhary, C. Mol Cell Proteomics 2012, 11, 1578-1585. (13) Manza, L. L.; Stamer, S. L.; Ham, A. J.; Codreanu, S. G.; Liebler, D. C. Proteomics 2005, 5, 1742-1745. (14) Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Nat Methods 2009, 6, 359-362. (15) Geiger, T.; Wehner, A.; Schaab, C.; Cox, J.; Mann, M. Mol Cell Proteomics 2012, 11, M111 014050. (16) Ostasiewicz, P.; Zielinska, D. F.; Mann, M.; Wisniewski, J. R. J Proteome Res 2010, 9, 3688-3700. (17) Soufi, B.; Kelstrup, C. D.; Stoehr, G.; Frohlich, F.; Walther, T. C.; Olsen, J. V. Mol Biosyst 2009, 5, 1337-1346. (18) Erde, J.; Loo, R. R.; Loo, J. A. J Proteome Res 2014, 13, 1885-1895. (19) Nel, A. J.; Garnett, S.; Blackburn, J. M.; Soares, N. C. J Proteome Res 2015, 14, 1637-1642. (20) Chiva, C.; Ortega, M.; Sabido, E. J Proteome Res 2014, 13, 3979-3986. (21) Wisniewski, J. R. Anal Chem 2016, 88, 5438-5443. (22) Udeshi, N. D.; Mertins, P.; Svinkina, T.; Carr, S. A. Nat Protoc 2013, 8, 1950-1960. (23) Vizcaino, J. A.; Csordas, A.; del-Toro, N.; Dianes, J. A.; Griss, J.; Lavidas, I.; Mayer, G.; Perez-Riverol, Y.; Reisinger, F.; Ternent, T.; Xu, Q. W.; Wang, R.; Hermjakob, H. Nucleic Acids Res 2016, 44, D447-456. (24) Kall, L.; Canterbury, J. D.; Weston, J.; Noble, W. S.; MacCoss, M. J. Nat Methods 2007, 4, 923-925. (25) Spivak, M.; Weston, J.; Bottou, L.; Kall, L.; Noble, W. S. J Proteome Res 2009, 8, 3737-3745. (26) Taus, T.; Kocher, T.; Pichler, P.; Paschke, C.; Schmidt, A.; Henrich, C.; Mechtler, K. J Proteome Res 2011, 10, 5354-5362. (27) Glatter, T.; Ludwig, C.; Ahrne, E.; Aebersold, R.; Heck, A. J.; Schmidt, A. J Proteome Res 2012, 11, 5145-5156. (28) Vandermarliere, E.; Mueller, M.; Martens, L. Mass Spectrom Rev 2013, 32, 453-465.

ACS Paragon Plus Environment

Page 10 of 17

Page 11 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure Captions Figure 1. Overview of sample preparation procedure. Cells were lysed and proteins were digested by LFASP. The digests were fractionated by bRP liquid chromatography, bRP fractions were pooled to generate five final bRP fractions, and K-ε-GG peptides were enriched by IAP. The original bRP fractions pooled are depicted with the same color and numbered according to the number of the final bRP fraction. Preparatory steps (i.e., passivation of ultrafiltration units and cross-linking of the anti-K-ε-GG antibody to protein A agarose beads) have been omitted for clarity (see the text for details). Samples analyzed by LC-MS/MS (single-shot proteomic analysis, Figure 2; analysis of K-ε-GG peptides, Figures 3 and S1; and comprehensive proteomic analysis -25 bRP fractions-, Figures 4 and S3), are indicated with an asterisk. bRP-LC, basic pH reversed phase liquid chromatography; LFASP, large-scale filter-aided sample preparation; IAP, immunoaffinity precipitation; Ub, ubiquitin. Figure 2. Efficiency, robustness and reproducibility of LFASP. Jurkat cells from three independent experiments (with ten replicate samples each) were lysed and digested using LFASP. Single-shot proteomic analysis was carried out using two µg of digest. Cleavage efficiency is represented by the percentage, based on the number (A) or abundance (B), of fully cleaved peptides (i.e., zero missed cleavages). The results of each experiment are expressed as the mean ± SD of ten replicate digestions. The mean ± SD of the number of peptides identified per replicate in each experiment is indicated in parentheses at the top. The quantitative reproducibility of peptide and protein abundances within each experiment was evaluated using the software Progenesis QI for proteomics. The distributions of the coefficients of variation of peptide and protein abundances (C) and the correlations (R2) between individual replicates of log2 peptide and protein abundances (D) are depicted (n = 7-10). Boxes represent the median and interquartile ranges (IQRs), and whiskers extend to 1.5 x IQR. The number of peptides and proteins used to evaluate the quantitative reproducibility are indicated in parentheses. Peptides and proteins with missing values were excluded from the correlation analysis. Figure 3. Benchmarking LFASP for ubiquitinome studies. Sample digests were fractionated by bRP, subjected to K-ε-GG enrichment and analyzed by LC-MS/MS (see Figure 1). Cleavage efficiency of LFASP (this study) is compared with state-of-the-art ubiquitinome studies that used urea-based in-solution digestion9,10. Cleavage efficiency is represented by the percentage, based on the number (A) or abundance (B), of peptides that are fully cleaved (i.e., zero missed cleavages for non-K-ε-GG peptides and zero or one missed cleavages for peptides with one K-ε-GG site). Peptides with two or three K-ε-GG sites only represent 1-3 % of the total number of K-ε-GG peptides and have been omitted from the figure for clarity. The complete comparison of the three datasets is provided in Table S2. Figure 4. Abundance-based distribution of miscleaved peptides in comprehensive proteomic analysis. Comprehensive proteomic analysis was carried out by LC-MS/MS analysis of the 25 bRP fractions collected (see Figure 1). All peptides quantified are binned based on their abundance in the digest. The percentage of fully cleaved and miscleaved peptides is shown for each bin. The distribution of peptides in bins is depicted with red circles.

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only

ACS Paragon Plus Environment

Page 12 of 17

Page 13 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 1

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2

ACS Paragon Plus Environment

Page 14 of 17

Page 15 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 3

ACS Paragon Plus Environment

Analytical Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4

ACS Paragon Plus Environment

Page 16 of 17

Page 17 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Figure 2. Efficiency, robustness and reproducibility of LFASP. Jurkat cells from three independent experiments (with ten replicate samples each) were lysed and digested using LFASP. Single-shot proteomic analysis was carried out using two µg of digest. Cleavage efficiency is represented by the percentage, based on the number (A) or abundance (B), of fully cleaved peptides (i.e., zero missed cleavages). The results of each experiment are expressed as the mean ± SD of ten replicate digestions. The mean ± SD of the number of peptides identified per replicate in each experiment is indicated in parentheses at the top. The quantitative reproducibility of peptide and protein abundances within each experiment was evaluated using the software Progenesis QI for proteomics. The distributions of the coefficients of variation of peptide and protein abundances (C) and the correlations (R2) between individual replicates of log2 peptide and protein abundances (D) are depicted (n = 7-10). Boxes represent the median and interquartile ranges (IQRs), and whiskers extend to 1.5 x IQR. The number of peptides and proteins used to evaluate the quantitative reproducibility are indicated in parentheses. Peptides and proteins with missing values were excluded from the correlation analysis. 159x102mm (283 x 283 DPI)

ACS Paragon Plus Environment