Quantification of Human Kallikrein-2 in Clinical ... - ACS Publications

Technical Note pubs.acs.org/jpr

Quantification of Human Kallikrein‑2 in Clinical Samples by Selected Reaction Monitoring Á kos Végvári,*,† Karin Sjödin,† Melinda Rezeli,† and György Marko-Varga†,‡ †

Clinical Protein Science & Imaging, Biomedical Center, Department of Measurement Technology and Industrial Electrical Engineering, Lund University, BMC C13, 221 84 Lund, Sweden ‡ First Department of Surgery, Tokyo Medical University, 6-7-1 Nishishinjiku Shinjiku-ku, Tokyo, 160-0023 Japan S Supporting Information *

ABSTRACT: Recently, the number of mass spectrometry-based quantification assays has been increased, partially due to the global efforts of chromosome-centric human proteome project (C-HPP). Our goal at the Chromosome 19 Consortium is to provide novel selected reaction monitoring (SRM) assays of proteins coded on chromosome 19. We have selected the two most useful signature peptides (NSQVWLGR and HNLFEPEDTGQR) of human kallikrein-2 (hK2 − NX_P20151) and developed an SRM assay. Details of the analytical parameters, including multiple transitions by peptides, are presented. The endogenous levels of hK2 were determined in clinical samples (n = 35). The limit of quantification was also estimated by spiking heavy isotope-labeled peptides into seminal plasma samples at various concentrations (LOQ ≈ 29 ng/ mL). KEYWORDS: chromosome 19, human kallikrein-2, selected reaction monitoring, mass spectrometry that of PSA in serum,7,8 which may provide independent diagnostic importance. Kallikrein-2 was shown to activate proPSA,9−11 pro-hK2,12 and also the zymogen form of urokinasetype plasminogen activator (uPA), an extracellular protease correlated with the aggressiveness of prostate cancer and metastasis.11,13 Additionally, hK2 has been revealed to cleave seminogelin 1 and 2 (SEMG1 and SEMG2)14 and possibly has a physiological role in the regulation of PSA activity. In the current stage, hK2 is not commercially available as an endogenous product but in multifold as tagged recombinant proteins at different sources, which poses a critical challenge in the design and procedure, whereby hK2 SRM assays for clinical use are being developed.15,16 To identify and quantify hK2 in biological samples, we have developed an SRM assay that is based on the two most suitable tryptic signature peptides, which is in accordance with the CHPP guidelines, especially in cases where peptides are needed for novel gene annotations.2 Our goal was to establish such a quantitative method using heavy isotope-labeled synthetic peptides and demonstrate its applicability in seminal plasma samples. We have successfully quantified hK2 in individual clinical samples (n = 35) as well as evaluated the analytical performance of the assay.

1. INTRODUCTION The cluster of kallikrein genes are localized on chromosome 19 (positions: 19q13.33 and 19q13.41), including well-characterized proteins such as prostate specific antigen (PSA) and kallikrein-2.1 Chromosome 19 proteins are investigated for identification and quantification in biological samples following general guidelines as defined by the global initiative of Chromosome-based Human Proteome Project (C-HPP).2 One of the intensive efforts made in the C-HPP is the development of suitable targeted proteomic assays for quantification, such as selected reaction monitoring (SRM). Human kallikrein-2 (hK2), also known as glandular kallikrein-1 (hGK-1) or tissue kallikrein-2, is a glycoprotein with serine-type endopeptidase activity (EC 3.4.21.35) belonging to the peptidase S1 family and the kallikrein subfamily.3 The zymogen form of hK2 is expressed as a single chain, a 261 amino acid long polypeptide, which is shortened upon activation with the signal peptide (1−18) and the propeptide (19−24), resulting in a mature hK2 form at 237 amino acid length. The sequential similarity between hK2 and PSA is 77% (identity in 201 amino acids).4,5 As a serine protease, hK2 preferentially cleaves the Arg-|-Xaa bonds in small molecular substrates and highly selectively releases Lysbradykinin (kallidin) from kininogen.3 Kallikrein-2 is expressed predominantly in the prostatic epithelium, which has been shown to be elevated in cases of prostate carcinoma, compared with normal or benign prostate tissues.6 However, the distribution profile of hK2 differs from © 2013 American Chemical Society

Received: May 4, 2013 Published: August 26, 2013 4612

dx.doi.org/10.1021/pr400423k | J. Proteome Res. 2013, 12, 4612−4616

Journal of Proteome Research

Technical Note

Table 1. Transitions of hK2 Peptides Used in the MRM Assay [M+H+]+ mass

precursor mass

charge state

fragment ion

fragment mass

charge state

NSQVWLGR

959.506

480.26

++

NSQVWLGR

969.514

485.26

++

1442.666

721.84

++

481.56

+++

726.84

++

484.90

+++

y7 y6 y5 y4 y7 y6 y5 y4 y10 y9 y8 y7 y4 y7 y6 y5 y10 y9 y8 y7 y4 y7 y6 y5

845.46 758.43 630.37 531.30 855.47 768.44 640.38 541.31 1191.84 1078.48 931.41 802.37 461.25 802.37 705.32 576.27 1201.57 1088.49 941.42 812.25 471.25 812.38 715.32 586.28

+ + + + + + + + + + + + + + + + + + + + + + + +

peptide

HNLFEPEDTGQR

HNLFEPEDTGQR

1452.674

2. MATERIAL AND METHODS

2.3. Sample Preparation

2.1. Biological Samples

Prior to analysis, the samples were thawed on ice and the protein content of the seminal plasma was determined by the Bradford method20 (BioRad, Hercules, CA). A volume (9−26 μL) corresponding to 0.2 mg protein was diluted with 6 M urea in 50 mM ammonium bicarbonate to a final volume of 200 μL (1 mg/mL). Axygen Maximum Recovery low-binding sample tubes and pipet tips were used throughout the experimental procedures. The samples were reduced with 2 μL of 1 M dithiothreitol for 1 h at 37 °C and alkylated with 10 μL of 1 M iodoacetamide for 30 min at room temperature in dark. To remove denaturant, buffer exchange was performed with 50 mM ammonium bicarbonate using an YM-10 Microcon centrifugal filter unit (Millipore, Billerica, MA). Tryptic digestion was achieved by adding 1 μg sequencing grade trypsin (Promega, Madison, WI) and incubating at 37 °C for 17 h on a block heater with shaking at 900 rpm. The reaction was stopped by the addition of 10 μL of 10% formic acid. The resulting protein digests were dried on speed vacuum centrifugation and restored in 50 μL of 5% ACN with 0.1% formic acid and stored at −20 °C until analysis. At the time of analysis the seminal samples were spiked with 5 fmol/μL of the heavy isotope labeled hK2 peptides and diluted 10-fold in 5% ACN with 0.1% formic acid.

Seminal plasma was prepared from semen obtained from young men undergoing investigation for infertility prior to final diagnosis of disorders (n = 30) and healthy volunteers (n = 5), following the guidelines of the Helsinki Declaration, as previously described.17 The collection of semen was approved by the ethical board at Lund University (approval number: LU 532-03), and the samples were processed according to the WHO guidelines (WHO, 1999). 2.2. In Silico Selection of Signature Peptides

The theoretical digestion of the neXtProt entry NX_20151 was performed by the PeptideMass tool (available at the ExPASy Proteomics Server website, http://expasy.org/sprot/18) using the following settings: iodoacetamide as alkylation agent without oxidation on methionine and no miss-cleavage. The resulted tryptic peptides were investigated for uniqueness by blast search on the UniProtKB website (http://www.uniprot. org/uniprot/). Finally, a list of tryptic peptides was prepared filtering by size (7−26 amino acids) for synthesis at low purity with and without heavy isotope labeling and alkylation at cysteine residues (JPT Peptide Technologies, Berlin, Germany). Synthetic peptides were dissolved in 100 μL of 20% acetonitrile (ACN) to obtain improved solvation of hydrophobic peptides. For quantification, two heavy peptides isotopically labeled with 15N and 13C in lysine (Δmass = +8) and arginine (Δmass = +10) at purity higher than 97% (AQUA QuantPro quality, concentration precision equal or better than ±25% from Thermo Fisher Scientific, Ulm, Germany) were used. These heavy isotope-labeled peptides were spiked into the biological samples at known concentrations, and the ratio between endogenous (light) and internal standard (IS) peptide was used to estimate the concentration of hK2 in the samples.19

2.4. SRM Assay of hK2

During the method development the software tool of Skyline v1.221 (MacCoss Lab Software, Seattle, WA) was used exclusively. Peptide sequence list was prepared manually based on the selected proteotypic tryptic sequences. Primarily, high numbers of transitions, all possible b- and y-ions that matched the criteria (from m/z > precursor-2 to last ion-2, precursor m/z exclusion window: 20 Th), were selected for each peptide at both 2+ and 3+ charge states. Finally, the three to five most intense transitions were selected for each peptide 4613



Technical Note

poor intensities observed in addition to the difficulty to handle oxidized and nonoxidized forms of the peptide. In accordance with the C-HPP efforts toward ENCODE,22 the selected two tryptic peptides were found unique for hK2. Further investigation revealed that both sequences are present in three isoforms of hK2 (1−3) but not in isoform-4; in addition, no single nucleotide polymorphism (SNP) sites are located on the signature peptides. These properties ensure that the SRM assay is highly selective to identify and quantify endogenous hK2 in biological samples.

by manual inspection of the data in Skyline, and the scheduled transition list was created for the final assay at both doubly and triply charge states when it was applicable. (See Table 1.) 2.5. Mass Spectrometric Analysis

Tryptic peptide digests were injected (2 μL) onto a trap column (Easy Column C18-A1 5 μm, 2 cm × 100 μm, Thermo Fisher Scientific, Waltham, MA) and separated on an analytical column (15 cm × 75 μm, packed with ReproSil C18-AQ 3 μm, 120 Å particles from Dr. Maisch, Ammerbuch, Germany) using an Easy n-LC II system (Thermo Fisher Scientific, Odense, Denmark) at 300 μL/min flow rate. The mobile phases were A: 100% LC-MS purity water with 0.1% FA and B: 100% ACN with 0.1% FA. The peptides were eluted with a linear gradient starting with 10% B to 35% B in 45 min, followed by a 5 min linear gradient to 90% B and a column wash at 90% B for 5 min. A TSQ Vantage triple quadrupole instrument (Thermo Fisher Scientific, San Jose, CA) was used with the Flex ESIinterface and operated in selective reaction monitoring mode in positive polarity. The MS analysis was conducted with 1750 V spray voltage and 0 V declustering potential. The transfer capillary temperature was set to 270 °C, and tuned S-lens value was used. SRM transitions were acquired in Q1 and Q3 operated at unit resolution (0.7 fwhm); the collision gas pressure in Q2 was set to 1.2 mTorr. The cycle time was 2.5 s in both nonscheduled and scheduled methods providing 12−14 data points in each peptide peak. The optimize collision energy along the regression calculation specified to TSQ Vantage was used as determined by Skyline.

3.2. Analytical Performance

To characterize the chromatographic separation of hK2 peptides, we monitored their retention times in 35 seminal plasma samples and found that variances were 3.0 and 3.2% for NSQVWLGR (Rt = 16.05 min) and HNLFEPEDTGQR (Rt = 14.53 min), respectively. Reproducibility of quantification was conducted by running 13 randomly selected samples in technical triplicates. The concentrations of endogenous hK2 peptides were determined by integration of the peak area using the weighted average of all transitions calculated with Skyline (Supplementary Table 2 in the Supporting Information). The coefficient of variance ranged between 0.9 and 10.7%. (70% of all CV was below 4%.) The efficiency of the proteolytic cleavage of the selected signature peptides was tested in triplicated digestion performed with three individual seminal plasma samples. The variance of overall quantification was ranging between 5.5 and 19.2% with both sequences, indicating good reproducibility (Supplementary Figure 1 and Supplementary Table 3 in the Supporting Information). These satisfactory results triggered no further optimization of digestion conditions, which may be necessary in the case of multiplexed SRM assays. Linearity of the SRM assay was determined in seven dilution steps spiking the heavy labeled peptides into a pool of 12 seminal plasma samples. Each determination was repeated five times; then, the area ratios of IS and corresponding endogenous peaks were plotted against the spiked IS concentrations (Figure 1). The linear regression fitting resulted in R2 values higher than 0.99 within the investigated concentration range (0.03−30 fmol/μL). The LOQ of NSQVWLGR and HNLFEPEDTGQR was determined at CV < 20% to be 0.3 and 0.03 fmol/μL, respectively. This value of LOQ (28.7 ng/mL of hK2) was below the endogenous level of hK2 in seminal plasma.

2.6. Data Evaluation and Quantification of hK2

The raw files generated on the triple quadrupole mass spectrometer were imported to Skyline for data analysis. Quantification was based on the calculation of ratios between the corresponding endogenous and internal standard peak areas. Peak integration was automatically performed in Skyline using Savitzky−Golay smoothing, whereas all imported data were inspected manually to confirm the correct peak detection. Further statistical analysis was done using Microsoft Excel.

3. RESULTS AND DISCUSSION 3.1. Selection of Signature Peptides

Using the consensus sequence of hK2 (NX_P20151) for deriving the list of theoretical tryptic peptides and filtering by the inclusion and exclusion criteria described in the Material and Methods section, we could identify eight potential signature peptides. (See Supplementary Table 1 in the Supporting Information.) However, further selection by blast searching for uniqueness has revealed that IVGGWECEK is present in both PSA and hK2. The concentration of PSA is 1000 times higher than that of hK2 in seminal plasma, and thus IVGGWECEK could not be used for quantification of hK2. Following an initial round of experiments characterizing the unique hK2 peptides in terms of ionization and fragmentation using crude synthetic peptides, we could recognize NSQVWLGR and HNLFEPEDTGQR as the most suitable signature peptides (Table 1). These tryptic sequences were then successfully employed for selective quantification of hK2 in clinical samples by spiking their heavy isotope-labeled synthetic peptides into the digested seminal plasma. Notably, SLQCVSLHLLSNDMCAR, highlighted as the most useful signature peptide in the SRMAtlas, was not included due to

3.3. Levels of hK2 in Seminal Plasma

The endogenous levels of hK2 in the seminal plasma samples were calculated by taking the ratio between the peak areas of the light (endogenous) and heavy peptides and correlate to the concentration of the heavy peptides spiked into the samples. The endogenous levels in whole seminal plasma were calculated by adjusting for the dilution at sample preparation. The calculations of the hK2 levels in seminal plasma were completed for the two different peptides individually. The measured concentrations are presented in Supplementary Table 4 in the Supporting Information. The comparison of the determination of hK2 levels in seminal plasma displayed good correlation (see Figure 2 and Supplementary Table 4) in the Supporting Information. However, the absolute concentration values varied because the amount of spiked IS was unknown (±25% as the supplier specified). The correlation coefficient between the determinations was excellent with R2 values of 0.95. 4614



Technical Note

based quantification determinations and contribute to the goals of C-HPP.

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ASSOCIATED CONTENT

S Supporting Information *

Reproducibility of digestion performed in triplicates with three individual seminal plasma samples, summary of tryptic peptides of hK2 (P20151) used for MRM assay development, reproducibility of digestion performed in triplicates with three individual seminal plasma samples, and summary of hK2 quantification based on two tryptic peptides (NSQVWLGR and HNLFEPEDTGQR). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46-46-222 3721. Fax: +46-46-222 4521. E-mail: Akos. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Martin Hornshaw and Egon Rosén at Thermo Fisher Scientific for mass spectrometry support. We are grateful for funding support from the Swedish Research Council, Vinnova, Knut and Alice Wallenberg Foundation, Crafoord Foundation, and Carl Trygger Foundation.

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ABBREVIATIONS ACN, acetonitrile; C-HPP, chromosome-centric human proteome project; CV, coefficient of variance; SRM, multiple reaction monitoring; hGK-1, glandular kallikrein-1; hK2, human kallikrein-2; IS, internal standard; LOQ, limit of quantification; MS, mass spectrometry; PSA, prostate specific antigen; Rt, retention time; SEMG1, seminogelin 1; SEMG2, seminogelin 2; uPSA, urokinase-type plasminogen activator

Figure 1. Linearity of the SRM assay determined by using heavylabeled IS peptides spiked into a pooled seminal plasma sample at various concentrations (0.03−30 fmol/μL). The LOQ (CV < 20%) is indicated with arrow.

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REFERENCES

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Figure 2. Correlations between the determinations of hK2 in seminal plasma by two different peptides.

4. CONCLUSIONS In accordance with recent quantitative proteomics efforts, we have developed a novel SRM method for human kallikrein-2 based on systematic investigation and selection of the two most suitable signature peptides. Using this assay, endogenous expression of hK2 was determined in seminal plasma samples digested directly without further processing. The analytical performance of the developed SRM assay was demonstrated and proved to be robust with good analytical precision. We believe that the introduction of this assay facilitates further MS4615



Technical Note

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Quantification of Human Kallikrein-2 in Clinical ... - ACS Publications

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