Clinically relevant profiling of proteoforms in human tear fluid using chip-based capillary

[D. Lopez-Ferrer, R. Huguet, A. Krupke, C.-H. Chen, A. Paulus, P.P.M. Raus, V. Zabrouskov, A.F.R. Huhmer, P.D.E.M. Verhaert ASMS2017_3]

6.3.1 Abstract

With the aim to demonstrate the potential of capillary electrophoresis hyphenated to high- performant orbitrap mass spectrometry for monitoring lacrimal proteoforms, we designed an

experiment to combine a microfluidic chip (ZipChipTM_{) capable of highly efficient, and in very}

short time, electrokinetically driven separations [Redman et al., 2016] with a Q Exactive HFTM

quadrupole-orbitrap hybrid MS/MS instrument.

The same tear sample, which had been extensively top-down characterized in Chapter 6.2, was

applied to Schirmer strips, and eluted in aqueous solution and directly analyzed by capillary electrophoresis (CE) coupled to mass spectrometry in without further sample processing. We demonstrate that highly reproducible electropherograms can be generated using very small protein samples. These CE-separated tear proteoform MS profiles can be generated in less than 5 min, and, employing the database of 50 proteoforms identified previously, all these proteoforms in the tear sample profile can be readily identified.

6.3.2 Introduction

Currently, Dry Eye Disease (DED) is diagnosed either by subject self-reported history, or by measuring tear quantity using Schirmer strips. Here, we evaluate the use of an attractive instrument combination for top-down proteomics as clinically viable biomarker detection strategy for DED, and, by extension, various eye-related and other diseases. We propose the combination of Schirmer strips for sample collection, protein extraction, and CE-MS/MS as an accurate and sensitive way to identify proteoform biomarkers in tear fluid.

6.3.3 Materials and Methods Sample Preparation

Protein standards (Pierce™ Intact Protein Standard Mix; see Table 6.2) to evaluate protein separations in the microfluidic CE device (Fig. 6.14) were purchased from Pierce. Tears were purchased from Lee Biosolutions (Maryland Heights, MO, USA), Schirmer Tear Test strips from Clement Clarke International (Harlow, UK).

Test Method(s)

Samples were introduced via autosampler into a CE microchip (ZipChipTM, 908 Devices, Boston,

MA, USA) attached to the ESI-MS. MS was performed on a Thermo Scientific™ Q Exactive HF™ hybrid quadrupole-orbitrap MS.

A schematic of the instrumentation is shown in Fig. 6.15.

Fig. 6.15 (A) CE ZipChipTM_{; (B) Microfluidic architecture of ZipChip integrating capillary electrophoresis and}

electrospray mass spectrometry; (C) Picture of electrospray plume illuminated with green laser; (D) benchtop QExactive HF with ZipChip CE source and CE autosampler for automated injections.

Six proteins were selected that (1) evenly covered a MW range of 12kD – 66kD; (2) presented mostly clean, modification and adduct-free ESI spectra; and (3) the ESI charge state distributions of which covered a wide m/z range between 500-2000 (Fig. 6.15).

Fig. 6.16 Biopharma FinderTM software deconvoluted masses for of each of the six proteins using sliding window

deconvolution feature.

Data Analysis

Raw MS files were deconvoluted using BioPharma Finder™ Mass Informatics Platform (Thermo Fisher Scientific, San Jose, CA, USA) and deconvoluted masses were matched to a database populated with tear proteoforms that were identified previously using top down proteomics applying high resolution high mass measurement accuracy LC-MSMS-based approaches

6.3.4 Preliminary Results

Ten replicate injections were done subsequently to assess the robustness of the analytical setup (Table 6.2). Five representative runs are shown in Fig. 6.17.

Protein ID Accession number Time CV Intensity CV

Human IGF-1 P05019 1.68 1% 1.83E9 25%

E. coli Exo Klenow P00582 1.73 0% 2.5E7 16%

Bovine carbonic anhydrase II P00921 1.78 0% 2.4E9 18%

Human Thioredoxin Q99757 1.85 0% 3.4E9 10%

Streptococcus Protein G Q54181 1.90 0% 3.3E9 24%

Streptococcus Protein AG P02976 2.02 0% 1E9 39%

Table 6.2 Standard proteins and their CE migration time and raw MS intensity variation over 10 different analyses.

Fig. 6.17 Five representative electropherograms from 10 replicate injections of the Pierce™ Intact Protein Standard Mix

The tear sample analyses yielded highly reproducible CE-MS profiles (Fig. 6.18). It is clear that, in contrast to the standard peak analysis which showed 6 separate peaks on 6 (carefully selected) proteins, each electropherogram peak of the true biological sample contains multiple mass spectral peaks. As a matter of fact, previous analysis of the exact same sample indicated that the sample contains, at least, 50 different proteoforms. This is illustrated by the inserts in Fig. 6.18, which represent mass spectra across selected electropherogram peaks.

Matching the (high accuracy) mass measurements of the CE separated proteoforms with the database, which was populated with the proteoforms extensively analyzed from this sample before, enabled for a confident identification of a substantial number of proteoform peaks. Not surprisingly these contain some of the ‘usual suspects’, i.e. some of the typical and relevant lacrimal proteins. These are listed in Table 6.3.

Fig. 6.18 Six consecutive replicate injections from 5 µL of human tears as starting material, illustrating high reproducibility of analysis with real biological samples.

UNIPROT NAME FUNCTION [Literature source]

O75368 SH3 domain binding glutamic

acid-rich protein like Related to glutaredoxins and expressed in retina [Bowes Rickman et al., 2006]

P01037 Cystatin SN Cysteine proteinase inhibitor [Barka et al., 1991]

P05109 S100 calcium binding protein

A8 Proinflammatory protein with potential antimicrobial activity [Zhou et al., 2009]

P06702 S100 calcium binding protein

A9

Proinflammatory proteins with potential antimicrobial activity [Zhou et al., 2009]

P12273 Prolactin-induced protein Potent antiangiogenic, proapoptotic effects [Priyadarsini et

al., 2014]

P31025 Lipocalin 1-like 1; lipocalin 1

(tear prealbumin) Primary lipid binding protein in tears, overproduced in response to multiple stimuli including infection and stress

[Dartt, 2011]

Q9GZZ8 Lacritin Secretion modulation by lacrimal acinar cells [Samudrfe et al.,

2011]

Q9Y5E8 Protocadherin beta 15 Crucial role in development of retina [Alagramam et al.,

2001]

P01009-1 alpha1-antitrypsin (SERPINA1) Controls several types of chemical reactions by blocking

(inhibiting) activity of certain enzymes [Sen et al., 1988]

Q02505-1 Mucin-3a Maintaining homeostasis of wet ocular surface [Hodges and

Dartt, 2013]

Table 6.3 List of relevant proteins in eye health monitored as identified proteoform MS peaks by our ZipChip-MS platform

6.3.5 Discussion / Conclusion

Whereas the potential of (chip-based) capillary electrophoresis has been recognized for several years [Carrilho and Garcia, 2014], its practical implementation is only recently becoming commercially available (Redman et al., 2016). Coupled to performant orbitrap mass spectrometry, CE using microfluidic devices represents a very easy and versatile technology for top-down tear analysis, such as for rapid screening of potential disease biomarkers. The

combination of the ZipChipTM and Q Exactive HFTM instrumentation allows for extremely

sensitive assays using very little amount of sample. The ZipChip MS technology allows for the robust monitoring of more than 30 proteoforms in tears in merely 5 min. and using minimal sample material.

Our analyses, moreover, demonstrate that CE-MS is a highy reproducible and sensitive technology for intact protein (proteoform) identification and assaying.

6.3.6 References

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Barka T, Asbell PA, van der Noen H, Prasad A (1991) Cystatins in human tear fluid. Curr. Eye Res. 10: 25-34

Bowes Rickman C, Ebright JN, Zavodni ZJ, Yu L, Wang T, Daiger SP, Wistow G, Boon K, Hauser MA (2006) Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE. Invest. Ophthalmol. Vis. Sci. 47: 2305-2316.

Carrilho E, Garcia CD (2014) Instrumentation for capillary electrophoresis and microchip electrophoresis. Electrophoresis 35: 2067.

Dartt DA (2011) Tear lipocalin: structure and function. Ocul Surf. 9: 126-138.

Hodges RR, Dartt DA (2013) Tear Film Mucins: Front Line Defenders of the Ocular Surface; Comparison with airway and gastrointestinal tract mucins. Exp. Eye Res. 117: 62–78. Priyadarsini S, Hjortdal J, Sarker-Nag A, Sejersen H, Asara JM, Karamichos D (2014) Gross

cystic disease fluid protein-15/prolactin-Inducible protein as a biomarker for keratoconus disease. PLoS ONE 9: e113310.

Redman EA, Mellors JS, Starkey JA, Ramsey M (2016) Characterization of intact antibody drug conjugate variants using microfluidic capillary electrophoresis – mass spectrometry. Anal. Chem. 88: 2220-2226.

Samudre S, Lattanzio FA, Lossen V, Hosseini A, Sheppard JD, Mc Cown RL, Laurie GW, Williams PB (2011) Lacritin, a novel human tear glycoprotein, promotes sustained basal tearing and is well tolerated. Invest. Ophthalmol. Vis. Sci. 52: 6265–6270.

Sen D K, Sarin G S, Mathur M D (1988) Alpha-1 antitrypsin and serum albumin levels in tear fluid of healthy subjects and in persons with conjunctival diseases. Indian J. Ophthalmol. 36: 22-26.

Zhou L, Beuerman RW, Ang LP, Chan CM, Li SF, Chew FT, Tan DT (2009) Elevation of human alpha-defensins and S100 calcium-binding proteins A8 and A9 in tear fluid of patients with pterygium. Invest Ophthalmol Vis Sci. 50:2077-2086.

6.4 Evaluation of heat stabilization in human tear sample preparation for top-down