Simple Tip-Based Sample Processing Method for Urinary Proteomic

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Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis David J Clark, Yingwei Hu, Michael Schnaubelt, Yi Fu, Sean Ponce, Shao-Yung Chen, Yangying Zhou, Punit Shah, and Hui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05234 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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Analytical Chemistry

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Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis

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David J. Clark1, Yingwei Hu1, Michael Schnaubelt1, Yi Fu2, Sean Ponce3, Shao-Yung Chen3,

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Yangying Zhou1, Punit Shah1, and Hui Zhang1*

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1Department

of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21231

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USA 2The

Bradley Department of Electrical and Computer Engineering, Virginia Tech, Blacksburg,

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VA, 24060 USA 3Department

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of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21231 USA

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*Corresponding author: Dr. Hui Zhang at [email protected]

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Keywords

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Urine proteomics, Automation, Mass Spectrometry

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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

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ABSTRACT

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Mass-spectrometry based urinary proteomics is one of the most attractive strategies to discover

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proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological

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diseases involving the kidney, prostate, and bladder, however, interfering compounds found in

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urine necessitate sample preparation strategies that are currently not suitable for urinary

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proteomics in the clinical setting. Herein, we describe the C4-Tip method, comprising a simple,

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automated strategy utilizing reversed-phase resin tip-based format and “on-tip” digestion to

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examine the urine proteome. We first determined the optimal conditions for protein isolation and

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protease digestion on the C4-Tip using the standard protein bovine fetuin. Next, we applied the

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C4-Tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS,

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with identified proteins from the C4-Tip method displaying a similar distribution of gene ontology

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(GO) cellular component assignments compared to identified proteins from an ultrafiltration

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preparation method. Finally, we assessed the reproducibility of the C4-Tip method, revealing a

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high Spearman correlation R value for shared proteins identified across all tips. Together, we

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have shown the C4-Tip method to be a simple, robust method for high-throughput analysis of the

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urinary proteome by mass spectrometry in the clinical setting.

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INTRODUCTION

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Urine is considered an attractive sample source for diagnosis, prognosis, monitoring, or prediction

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of responses to treatments of urological diseases due to its proximity, availability and non-

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invasive, simple collection procedure. In additional, the biological function of the kidneys to filter

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circulating plasma allows for the abundance of proteins secreted into urine to reflect the systemic

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physiological status of the patient, such as in diabetes and heart diseases1. Dissimilar to plasma,

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which displays a dynamic range of protein concentration up to 10-orders of magnitude and is

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dominated by several highly abundant proteins (i.e. albumin, immunoglobulins, fibrinogen), the

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urine proteome is viewed with less dynamic range and is comprised of a smaller percentage of

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plasma-derived proteins making it an attractive human biological fluid for protein sourcing 2,3.

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In addition to proteins, the composition of urine includes urea, inorganic salts, and other

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biomolecules and compounds that can confound proteomic analyses. To address this, a variety

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of urine sample preparation methods have been explored such as protein precipitation,

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ultrafiltration, and analytical ultracentrifugation4. However, due to inherent drawbacks of each of

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the methods, with few exceptions5, these techniques have not been adapted for high-throughput

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urinary protein sample preparation which is necessary for clinical analysis.

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For mass spectrometry-based proteomic analysis, sample preparation includes protein extraction

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from the sample source, removal of contaminants, followed by protease digestion to generate

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peptide fragments. For high-throughput sample analysis, these steps must be optimized and then

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integrated to generate a method that is robust and reproducible. Previously, reversed-phase

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chromatography has been utilized to isolate small sets of urinary proteins6–8, but the reversed-

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phase matrix has not been fully explored in the context of urinary proteomics including protein

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isolation and protease digestion followed by mass spectrometry analysis. Although reversed-

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phase C-18 resin has routinely been used in the proteomics field for peptide binding, resin material 3 ACS Paragon Plus Environment


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with shorter alkyl chains (e.g. C4) shows better recovery of proteins relative to resin materials with

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longer alkyl chains (e.g. C8,C18)9. In this study, we evaluated the utility of an automated, tip-

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based format (C4-Tip) for urinary protein isolation using C4 reversed-phase resin paired with “on-

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tip” protease digestion and mass spectrometry to examine the urine proteome.

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EXPERIMENTAL SECTION

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C4-Tip Fabrication. A polyethylene sheet (2 mm diameter) was inserted into Thermo Scientific

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matrix D.A.R.T.s tip with a volume capacity up to 300 L. For C4-Tips, 30 mg of C4 reverse phase

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resin in 300 L of methanol was loaded into the tip, with a second polyethylene sheet (5 mm

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diameter) inserted to seal the resin material. The packed C4-Tips were then ready for use in the

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Versette Liquid Handler (Thermo Scientific).

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Isolation and protease digestion of urinary proteins using C4-Tip. Each liquid

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aspiration/dispense cycle was performed using 200 L of buffer in approximately 2 min at room

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temperature unless otherwise noted. C4-Tips were conditioned with 0.1% trifluoroacetic acid

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(TFA) in 50% acetonitrile (ACN), followed by 0.1% TFA (10 cycles each). Solubilized proteins

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were acidified with formic acid (pH < 3) and coupled to C4-Tips (15, 30, 60 cycles for Figure S1A

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and 60 cycles for rest of other experiments) in a total volume of 300 L. C4-Tips were rinsed with

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0.1% TFA and then 100 mM triethyl ammonium bicarbonate (TEAB) to remove unbound and

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contaminant material (10 cycles each). Bound proteins were reduced with 10 mM Tris 2-

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carboxyethyl phosphine (TCEP), and alkylated with 15 mM iodoacetamide (20 cycles each).

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Protease digestion was performed (1:40 enzyme/protein) in 50 mM TEAB and 30% ACN with 120

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cycles of aspirating/dispensing to recover digested peptides directly from C4-Tips to solution. C4-

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Tips were subsequently rinsed with 50% ACN, 0.1% TFA to recover remaining peptides. Peptides

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directly released from C4-Tips by protease digestion and peptides eluted by 50% ACN, 0.1% TFA

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were pooled, dried, reconstituted in 0.1% formic acid (FA), and subjected to C-18 clean-up via

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the StageTip method10. Desalted peptides were labeled with 10-plex Tandem-Mas-Tag (TMT)

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reagents following manufacturer’s instructions. Peptides were resuspended in 50 mM HEPES,

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pH 8.5 and labeled with TMT reagent resuspended in anhydrous acetonitrile for 1 h at RT. The

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reaction was quenched using 5% hydroxylamine at RT for 15 min, and differentially labeled

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samples combined. The pooled peptides were then subjected to clean-up using C-18 SepPak

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columns and dried down.

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RESULTS AND DISCUSSION

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Workflow of C4-Tips. To develop a sample preparation methodology that would be high-

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throughput for urinary protein extraction and digestion for MS-based proteomics, we devised a

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reversed-phase tip-based format using C4 resin. Previous reports have indicated the utility of “in-

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tip/on-tip” digestion methodologies11–13, but few have been adapted for urinary proteomic analysis

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due to the challenges inherent to urine’s unique composition. As illustrated in Figure 1, urine

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samples were acidified and proteins were bound to the C4-Tips using repeated

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aspiration/dispensing steps. After binding, proteins would be washed with 0.1% TFA to remove

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contaminants (Desalting), followed by 100 mM triethyl ammonium bicarbonate (TEAB) to adjust

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the pH in each of the tips. Proteins are then subjected to washes with Tris 2-carboxyethyl

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phosphine (TCEP) and iodoacetamide (IAA), followed by an additional 50 mM TEAB rinse step

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(Reduction & Alkylation). Proteins were then tryptically digested, releasing peptides directly from

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the C4-Tip, followed by elution of the remaining peptides with 50% ACN, 0.1% TFA. Two released

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peptide fractions were then pooled and then subjected to C-18 clean-up prior to nano-ESI-LC-

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MS/MS.

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Assessment of binding time for protein on C4-Tips. To determine the amount of time

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necessary for sufficient binding of proteins from solution to C4-Tips, we utilized the standard 5 ACS Paragon Plus Environment


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protein fetuin and measured unbound protein material via BCA Assay. As seen in Figure S1A,

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300 g of fetuin in 250 l of 0.1% TFA was aspirated/dispended through the C4-Tips and

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remaining protein in the solution was measured at 30 min (15 cycles), 1 h (30 cycles), and at 2 h

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(60 cycles). We observed approximately 80% of the protein binding after 30 min, with 90% bound

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after 2 h. This results mirror a previous study, showing rapid adsorption of proteins onto C4

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material after 30 min, and adsorption plateauing afterwards when evaluated over 24 h14,

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suggesting that 2 h was sufficient for protein binding. We next evaluated the maximum capacity

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of the C4-Tip by binding various amounts of fetuin (100-600 g) onto a C4-Tip for 2 h, and

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measuring the amount of unbound material (Figure S1B). Our results indicated that 30 mg of the

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C4 resin could bind almost 400 g of protein material. The protein concentration of normal urine

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is estimated to be between 0-14 mg/dL (0-140 g/mL)15, and the binding capacity of the C4-Tip

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would be sufficient to allow for direct analysis of urine; even in cases of increased protein urine

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concentrations (proteinuria)16. Next, we examined the impact of serial binding of protein samples

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on the C4-Tips. We bound 100 g, 150 g, or 300 g of fetuin in three, two, and one periods of

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sample binding (each period consisted of a total of 2 h (60 cycles)), respectively, observing almost

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90% of material bound (Figure S1C). Finally, we assessed the amount of material eluted from

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the C4Tips. After washing the C4-Tips with 0.1% TFA, proteins were eluted with 50% ACN, 0.1%

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TFA. BCA Assay was used to measure the recovery of the fetuin indicating 50% protein of protein

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was released from the C4-Tips (data not shown). This latter observation suggested protein

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material remained bound on the C4 resin after elution and prompted us to consider an alternative

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method that would yield equivalent or better sample recovery, and also further simplify our sample

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preparation method.

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“On-Tip” Protein Digestion. We next wanted to explore utilizing an “on-tip” automated digestion

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format via the C4-Tips for processing protein samples. Protein sample preparation for tryptic


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digestion generally involves a reduction step, to break the disulfide bonds of proteins, followed by

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alkylation, to prevent the reforming of the disulfide bonds, with the inclusion of these steps

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improving protease cleavage and sequence coverage17. Following fetuin protein binding to the

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C4-Tips (2 h), reduction and alkylation was carried out (40 min each) prior to protease digestion.

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To determine the optimal buffer for “on-tip” protein digestion and sample recovery, we examined

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a total of five buffer compositions. Following protein digestion (6 h total), recovered peptides were

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then analyzed via mass spectrometry. The entire automated C4-Tip procedure (Figure 1) could

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be performed in a total of 9 hours. We observed the buffer composition influenced the recovery

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of peptides, as well as the efficiency of protease cleavage of fetuin (Table S1). Inclusion of 30%

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ACN in our digestion buffer not only resulted in a higher recovery of peptides more consistently,

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but also greatly improved the efficiency of trypsin enzyme activity as indicated by the reduced

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missed cleavage rate (Table S1). It has been previously shown that the presence of ACN can

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serve to denature a protein18, and protein digest of individual proteins have been carried out in

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high organic buffers with improved peptide recovery19. We found higher organic composition (50%

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ACN) to be more varied in peptide recovery and digestion efficiency, possibly due to the buffer

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composition changing as the organic solvent evaporated during the digestion step. With these

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results, we decided to apply our C4-Tip methodology with 50 mM TEAB buffer with 30% ACN to

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examine the proteome of normal urine.

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Comparison of C4-Tip digestion. We then compared the C4-Tip method to other methods of

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sample preparation of urine for proteomic analysis. Two 25 mL aliquots of normal urine were

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processed using a concentrator unit with 10 kDa mass cut-off membrane. The volumes for both

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were reduced 40-fold. From one aliquot, approximately 300 g (1g/L) of protein from the

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concentrated urine sample was acidified and bound to the C4-Tip. The second aliquot was buffer

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exchanged (BE) into 50 mM Tris-HCl, pH 7.5, which allowed for a protein concentration

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measurement that could then be extrapolated to the non-buffer exchanged sample (concentrated 7 ACS Paragon Plus Environment


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sample). SDS-PAGE analysis indicated a similar protein profile in each urine preparation (Figure

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S2A). As previously noted, there are a variety of preparation methods for evaluating urine protein

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composition. Ultrafiltration is a useful technique as it facilitates sample volume concentration,

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while simultaneously removing salts and other molecules4, in addition to allowing the exchange

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of the urine sample into a digestion compatible buffer. To determine whether the C4-Tip method

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was a comparable method of urine sample preparation, we examined the performance between

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ultrafiltration and our described technique, utilizing the buffer exchange (BE) urine aliquot and

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concentrated urine aliquot, respectively. In parallel, 300 g (1g/L) of BE urine was bound to a

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C4-Tip, in addition to a 200 g aliquot of BE urine subjected to in-solution digest in two conditions

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– 8M urea and 30% acetonitrile (ACN). In assessing these four digestions conditions, we could

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evaluate the performance of the C4-Tip compared to the well-established method of ultrafiltration

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followed by in-solution digestion, as well as determining any bias that may be introduced by the

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C4-Tip format, including the C4 matrix as well as the digestion buffer composition. Analyzing ~1

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g of peptide material via nano-ESI-LC-MS/MS, using the C4-Tip format we identified 813

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proteins groups from the concentrated urine sample and 725 from the BE urine sample,

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respectively; 787 protein groups from the in-solution digest of the BE urine sample, and 807

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protein groups from the 30% ACN in-solution digest of the BE urine sample. When comparing the

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gene annotations of the proteins identified between the concentrated urine sample digested via

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the C4-Tip and BE urine sample digested in-solution, we observed 547 proteins commonly

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identified between the two methods (Figure S2B). Overall, we observed an overlap range of 54-

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62%, depending on the total number of proteins identified in the respective four conditions, with

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108, 27, 108, and 59 uniquely identified proteins in the C4-Tip, C4-Tip BE, In-solution BE, 30%

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ACN BE digests, respectively. Utilizing PANTHER cellular component gene ontology (GO)

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assignments20, the identified proteins from the individual sample digestions displayed a similar

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distribution of classified proteins regardless of the sample preparation methodology (Figure S3).


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Previously, it has been shown the method of urinary protein extraction can impact both yield and

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proteins recovered21, however the overlap of identified proteins and the result of our GO analysis

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indicate an equivalent representation of the urine proteome regardless of the digestion method

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employed.

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In order to assess the distribution of individual protein abundance in each of the four digestion

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conditions, we utilized the intensity-based absolute quantitation (iBAQ) algorithm that is part of

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MaxQuant software suite 22–24. We found a similar dynamic range of protein abundance between

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all three sample preparations, spanning ~5-orders of magnitude (Figure 2) which is similar to

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another report utilizing a filter plate-based method5. Highly abundant proteins observed across all

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four preparations, many of which are glycoproteins, included AMBP, ALB, CD59, UMOD, PTGDS

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and immunoglobulins IGKC and IGHA1 and are in agreement with other studies relating overall

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urine protein abundance (Table S2)25. In our iBAQ results, we did not observe albumin as the

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most abundant protein in the urine proteome in any of the three preparations as reported

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elsewhere25,26. Although we would have to consider the systemic impact of variant digestion

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methods employed (e.g. C4 resin hydrophobicity, membrane absorption, protein solubility), the

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reported iBAQ values for the top urine protein and albumin were proportional between the C4-Tip

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method and buffer-exchanged in-solution digestion method, and without a calibration curve of

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protein/peptide standards to ascertain accurate protein abundance, the use of iBAQ to infer

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proportional abundance could inaccurately report specific protein abundance within a sample27.

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Other studies have utilized a lectin-affinity approach to circumvent the challenge of serum protein

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abundance (e.g. albumin) and identify glycoproteins of interest in the urine proteome28, however,

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our method enables a global protein profiling of urinary samples, and would be applicable for

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monitoring albuminuria resulting from chronic kidney diseases29. Inherent to our described

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method is the potential loss of endogenous urine peptides due to the use of C4 for protein-level

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binding, which is not optimal for peptide-level binding. Although this was not explored in our 9 ACS Paragon Plus Environment


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current investigation due to the unique challenges of peptidome data acquisition and bioinformatic

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analysis30, subsequent studies can explore the utility of the C4-Tip method for protein-depletion

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prior to direct peptidome profiling of urine.

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Next, we employed label-free quantitation (LFQ) using MaxQuant, to quantify the relative

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abundance of proteins across all four sample preparation conditions. MaxQuant LFQ intensity

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values were reported for 340 proteins across all methods, with reported Spearman correlation R

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values being highest between the two C4 Tip digestion conditions (R = 0.932) (Figure S2C). The

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correlation values between the C4 Tip and in-solution and 30% ACN digestions was 0.778 and

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0.802, respectively, which is comparable to previous report exploring protein-level correlation of

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digestion replicates utilizing either an in-solution or FASP-based sample preparation strategy31,32.

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Overall, these results indicate the performance of the C4-Tip is comparable to the previously

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established method of ultrafiltration followed by in-solution digestion for examining the urinary

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proteome.

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Reproducibility of C4-Tips. To assess the reproducibility of the C4-Tip format, three C4-Tip

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digests were performed using normal, concentrated urine. In total, 948 protein groups were

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identified across all three C4-Tip digestions, with 814, 790, and 801 protein groups identified from

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Tip 1, Tip 2, and Tip 3 digestions, respectively. (Figure S4A). In total, almost 70% of the proteins

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were commonly identified in all three tips, with differences in identified proteins most likely related

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to the biases of data-dependent MS2 analyses33,34. In this dataset, we observed an average of 70%

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of peptides having no missed cleavages, which is comparable to other studies reporting missed

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cleave rates ranging from 30-40% using in-solution digestion 31,32. Using the LFQ Intensity values

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reported for 614 protein groups identified across all three tips and we assessed the reproducibility

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of the C4-Tip method. The Spearman correlation R values ranged from 0.975 to 0.978 among the

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C4-Tip replicates, revealing excellent reproducibility across all three C4-Tips (Figure S4B and 10 ACS Paragon Plus Environment

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S4C). In addition, we observed a similar distribution of the LFQ log intensity between all three

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tips, when plotted against total protein number (Figure S4D). Taken together, these results

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indicate the C4-Tip method displays a high level of tip-to-tip repeatability for the analysis of the

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urinary proteome.

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Application of C4-Tip to urinary proteomics. Next we applied the C4-Tip method to directly

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analyze urine specimens from healthy donors (Table S3). Three 300 L volumes of urine were

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removed from each sample and subjected to the C4-Tip methodology. Following protein digestion,

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we determined the starting protein concentration of each urine sample and the recovered amount

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of peptide material for each of the C4-Tips (n = 3 for each sample) using a Bradford Assay. We

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observed variable protein concentrations for each of the urine samples, and also measured

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variable digestion efficiencies of certain samples (Figure S5A). Several factors that may have

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contributed to this observed variability of sample recovery could include the starting protein

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concentration, which would have impacted the total amount of protein digested, or the amount of

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insoluble material present in the individual urine sample. In addition, the manual construction of

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the C4-Tips could be variable in itself and influence the recovery of peptide material, however,

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this could be addressed by incorporating appropriate QC metrics. Regardless, for all of the

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samples we found that we had sufficient peptide material (> 1 g) for TMT-labeling and mass

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spectrometry analysis, and devised a TMT-labeling strategy that would enable us to determine

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sample preparation reproducibility, in addition to determining sample labeling accuracy (Figure

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S5B). Two TMT sets were generated, and subjected to mass spectrometry analysis, wherein we

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identified 1150 and 1032 protein groups, respectively, which is comparable to the average number

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of urinary proteins identified without fractionation35. Utilizing the reported TMT intensities for each

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of the proteins, we were able to calculate the correlation of protein abundance in each of the

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sample preparations, observing values greater than 0.91 for all technical replicates (Figure 3;

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Table S4), indicating a high level of reproducibility irrespective of the variability of measured urine



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protein concentration and amount of recovered peptide material. This is a promising result in the

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context of urinary proteomics, as the protein concentration and composition of urine is extremely

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variable, and our results suggest the C4-Tip methodology sufficiently binds enough urinary protein

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material even from samples with extremely low protein concentrations (e.g. 0.017 g/mL) for

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downstream proteomic analysis. Alternatively, for urine samples with extremely low protein

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concentration, serial loading (Figure S1C) could be employed to maximize sample binding and

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recovery. Overall, results mirror our previous analysis using LFQ to assess tip-to-tip repeatability,

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and show that the automated C4-Tip methodology can facilitate direct analysis of urine proteome

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without significant pre-processing steps.

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CONCLUSION

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With its non-invasive and simple collection procedures, urine is considered an ideal biological

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fluid for disease monitoring and diagnosis. However, the composition of urine requires extensive

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sample preparations strategies prior to proteomic analyses, many of which are difficult to adapt

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for high-throughput analysis in the clinical setting. Reversed-phase chromatography has been

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utilized for analyzing small sets of urinary proteins, but we sought to adapt this technique into an

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automated, tip-based format. First, we evaluated our technique using a standard protein to

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determine optimized protein binding and digestion conditions. Next, we compared the C4-Tip

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format to the established technique of ultrafiltration followed by in-solution digestion for examining

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the urinary proteome, revealing that the C4-Tip is comparable to the previously used technique.

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We then examined the reproducibility of the C4-Tip format for analyzing the urine proteome,

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revealing a high level of repeatability between individual C4-Tip experiments. Finally, we applied

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the C4-Tip method to enrich and digest urinary proteins from unprocessed urine, indicating that

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the C4-Tip method is a robust methodology for urinary proteomics. Overall, we have shown the

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C4-Tip format is a simple, reproducible technique for proteomic sample processing, and can have

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applications in the clinical setting for investigating the urinary proteome, as well as being

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expanded to include other biological samples. 12 ACS Paragon Plus Environment

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SUPPORTING INFORMATION

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Extended Methods Section: Chemicals and Materials, Information related to in-solution digestion,

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Nano-ESI-LC-MS/MS Analysis, and Data Analysis for Protein Identification and Quantification

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(PDF)

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Figures: Bar graphs of C4-Tip binding time and capacity; overlap of protein identifications between

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disparate digestion methods of urinary proteomics; PANTHER GO assignments of protein

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identifications between disparate digestion methods of urinary proteomics, reproducibility of C4-

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Tip digestion method; direct analysis of urine proteome using C4-Tip TMT labeling schematic and

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bar graph of measured urine peptide recovery (PDF)

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Tables: Measured peptide recovery and peptide missed cleavages rates in various C4-Tip

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digestion buffers; Top 20 abundant proteins from disparate digestion methods; Normal urine

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sample information utilized for direct analysis of the C4-Tip methodology; Reported Pearson

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correlation values for direct analysis of normal urine samples; C4 Tips MaxQuant search results;

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C4 Tip BE MaxQuant search results; In-solution BE digestion MaxQuant search results; 30% ACN

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in-solution BE digestion MaxQuant search results; PANTHER Gene Ontology (GO) Assignment

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Results for disparate digestion methods; Direct Analysis of normal urine Proteome Discovered

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2.2 search results (including identified proteins, reported TMT ratios); TMT plex #1; Direct

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Analysis of normal urine Proteome Discovered 2.2 search results (including identified proteins,

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reported TMT ratios); TMT plex #2 (XSXL)

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ACKNOWLEDGEMENTS

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This work was supported by the National Institutes of Health, National Cancer Institute, the Early

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Detection Research Network (EDRN, U01CA152813), the Clinical Proteomic Tumor Analysis

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Consortium (CPTAC, U24CA210985). The authors declare no competing conflicts of interest.

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Figure 1. Urinary proteomics using C4-Tips. Protein sample is acidified and proteins are bound onto the C4-Tips. Next, tips are washed and bound proteins subjected to reduction, alkylation, and “on-tip” protease digestion. Protease digested peptides are released directly during digestion, and remaining peptides were recovered via 50% ACN washing and elution. Both fractions are pooled, and peptides are analyzed via LC-MS/MS.

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Figure 2. Dynamic range of protein abundance of the urine proteome using disparate digestion methods. Reported iBAQ values for proteins identified using the C4-Tip digestion 18 ACS Paragon Plus Environment

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format (A), the C4-Tip buffer exchanged digestion (B), in-solution digestion (C), and in-solution digestion containing 30% acetonitrile (D), respectively. iBAQ values were plotted against the protein’s abundance rank.

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Figure 3. Direct analysis of urine protein using the C4-Tip digestion method Heat map of correlation coefficients using reported TMT protein intensities shows high reproducibility of the technical replicates for the five normal urine samples. Pearson correlation analyses revealed R value of > 0.90.

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Simple Tip-Based Sample Processing Method for Urinary Proteomic

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