Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS

Chemicals and reagents

Human CYFRA 21-1 calibrator grade, containing 0.083 mg/mL CYFRA 21-1 and a total protein concentration of 33.9 OD280 units, was purchased from Meridian Life Science (Memphis, TN, USA). Monoclonal anti-cytokeratin 19 antibody (A53-B/A2) produced in mouse was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Stable isotopically labeled internal standard (IS) AALEDTLAETEA[R_¹³C₆, ¹⁵N₄] was obtained from Pepscan Presto BV (Lelystad, The Netherlands), dissolved in 10 % acetonitrile (ACN), 0.1 % formic acid (FA) and MilliQ at 900 ng/mL and stored in aliquots at −70 °C. Protein G (pG) Dynabeads and BS(PEG)₅ were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sequencing grade modified trypsin was purchased from Promega (Madison, WI, USA), diluted in 50 mM acetic acid to a concentration of 30 ng/μL, aliquoted and stored at −70 °C. LC-MS grade mobile phase solvents were obtained from Biosolve (Valkenswaard, The Netherlands). Cetuximab (Erbitux, Merck, Darmstadt, Germany) was buffer-exchanged to PBS pH 7.6 using an Amicon 50 kDa MWCO centrifugal filter (Merck Millipore) according to manufacturer’s instructions and the final protein concentration was checked (9.25 g/L). Adult bovine serum (BOV), purchased male AB serum (HUM-S) and all other reagents were obtained from Sigma-Aldrich (Saint Louis, MO, USA).

Antibody coupling to pG functionalized magnetic beads

The coupling procedure of the KS19.1 antibody to pG functionalized magnetic beads was performed multiple times in LoBind Eppendorf/Falcon tubes according to the following general procedure: 0.5 mg pG Dynabeads were washed twice in 400 µL conjugation buffer (100 mM sodium phosphate, 70 mM NaCl, 0.05 % Tween-20 (v/v), pH 8.0). The supernatant was removed and 4 µg KS19.1 antibody dissolved in conjugation buffer was added to the beads. After 30 min incubation with rotation, 25 µM BS(PEG)₅ in cold conjugation buffer was added and incubated for 30 min with rotation. The reaction was quenched by adding 50 µL of 1 M Tris-HCl, pH 8.0 for 15 min with rotation. The supernatant was removed and unbound antibody was eluted by adding 50 µL (elution 1) and 400 µL (elution 2) elution buffer (1 M NaSCN in 100 mM Tris-HCl, pH 7.5) for 5 min with rotation. The beads were then washed twice with 400 µL PBS, 0.1 % Tween-20, pH 7.6 and subsequently stored in this buffer at 4 °C until further use. For large-scale experiments, volumes were multiplied by the number of equivalents.

AS precipitation and IA purification of CYFRA 21-1

Serum samples were processed by subsequent protein precipitation and isolation of CYFRA 21-1 by IA purification. Protein precipitation was performed by slowly adding 1 mL of 4.1 mol/L AS to 1 mL serum in a 2 mL LoBind Eppendorf tube (50 % v/v AS). After 30 min incubation with rotation, the sample was centrifuged for 10 min at 13,400 rpm. The supernatant was discarded and the pellet was redissolved in 1 mL PBS, pH 7.6. IA purification was performed by adding 50 µL of the coupled beads to the dissolved pellet, which was then incubated for 1 h with rotation. Hereafter, the supernatant was removed and the beads were washed twice with 200 µL PBS, pH 7.6.

Tryptic digestion

The washed beads were shortly centrifuged to allow removal of all supernatant whereafter 33.1 µL digestion solution was added (31.9 µL 50 mM Tris-HCl, 1 mM CaCl₂ + 1 µL 1 M Tris-HCl, pH 8.0 + 0.2 µL IS per sample). The sample was denatured for 5 min at 95 °C at 1,200 rpm after which the mixture was cooled down and shortly centrifuged. Digestion was initiated by adding 300 ng trypsin for 1 h at 37 °C at 1,200 rpm. To stop the reaction, the mixture was put on ice and 6.9 µL ACN with 30 % FA was added. The sample was shortly centrifuged and transferred to a new LoBind Eppendorf tube, which was then centrifuged for 10 min at 13,400 rpm and finally transferred to a LC-MS vial.

Signature peptide selection

The tryptic peptides of in silico digested CYFRA 21-1 were evaluated based on uniqueness in the human genome using BlastP software (https://blast.ncbi.nlm.nih.gov/Blast.cgi), number of amino acids (6<n<25), presence of the unstable amino acids (M, C, N, Q) and signal intensity in mass spectrometry. Since the CYFRA 21-1 stock also contained other proteins, 10 µg CYFRA 21-1 was isolated by IA purification, followed by denaturation, digestion, and consecutive high resolution quadrupole time-of-flight (QTOF) LC-MS^e (Waters, Milford, MA) analysis to confirm sequence identity of the precursor and product ions. This data acquisition mode alternates low-energy and high-energy collision energies (CE), enabling the measurement of precursor and product-ions within one chromatographic run.

Instrumentation and chromatographic conditions

Signature peptide (SP) selection was performed in LC-MS^e mode on a Xevo G2 QTOF coupled to an Acquity UPLC I class binary solvent manager and Acuity UPLC Sample Manager-FL (Waters, Milford, MA, USA). Chromatographic separation was performed on an Agilent Polaris C18-A (2.0 × 150 mm, 3 µm) column with a flowrate of 0.3 mL/min and column temperature of 50 °C. The mobile phases consisted of (A) 0.1 % FA in MilliQ and (B) 0.1 % FA in ACN. The gradient in volume percentage of mobile phase B per minute was 5–60 % B (0.0–53.5 min), 60–80 % B (53.5–53.6 min), 80 % B (53.6–55 min), 80–5% B (55–55.1 min) and 5 % B (55.1–60 min). Electrospray ionization (ESI) was operated in positive mode. Mass spectra were collected in MS^e mode with ramped CE from 20 to 50 V over the range of 100–2000 m/z. Peptide sequencing was performed using the BioLynx application within the software (MassLynx 4.1, Waters, Milford, MA). Quantitative analysis was done on a LCMS-8045 triple quadrupole coupled to a Nexera-I LC-2040C 3D Plus (Shimadzu, Kyoto, Japan). Chromatographic separation was carried out using the same column, flow rate and column temperature as described above with a gradient in volume percentage of mobile phase B per minute of 5–60 % B (0.0–13.5 min), 60–80 % B (13.5–13.6 min), 80 % B (13.6–15.0 min), 80–5% B (15.0–15.1 min) and 5 % B (15.1–20 min). The ESI was operated in positive mode with the optimized transition settings of the SP and IS listed in Supplementary Table 1.

Assay calibration and validation

Calibration standards were prepared in BOV using 1.0, 1.6, 3.1, 6.3, 13, 25, 50 and 100 ng/mL CYFRA 21-1 and subsequently stored at −70 °C. For each analytical run, peak area ratios of the SP and IS were plotted vs. the nominal concentrations of the calibration standards using linear least-squares regression with a weighing factor of 1/x². The lower limit of quantification (LLoQ) was checked by comparing the peak area of the 1.0 ng/mL calibration standard with blank BOV samples (both n=4) at the retention time of the SP (LLoQ at least 10-fold higher compared to the blank). Quality control (QC) samples were prepared in BOV using CYFRA 21-1 concentrations of 1 ng/mL (LLoQ), 3 ng/mL (low), 40 ng/mL (medium) and 80 ng/mL (high). The within run and between run precision and overall accuracy were evaluated at the four QC levels by measuring five replicates over 3 days and analyzed using one-way analysis of variance according to CLSI protocol EP15-A2 [24]. Precision was expressed as coefficient of variation (CV, %) and the accuracy was expressed as percent bias (% bias). The results were compared to European Medicines Agency (EMA) guidelines, which recommend acceptance criteria for chromatographic methods of <15 %, with exception of the LLoQ, which should be <20 %. Carry-over was assessed by measuring blank BOV samples after the highest calibration standard (e.g. 100 ng/mL).

Collection of patient serum

Sera were selected from a clinical study (lung marker study NL9146, ICTRP Search Portal [who.int]) described elsewhere [11, 14, 20, 25, 26], ethically approved by the Medical Research Ethics Committees United (NL58985.100.16). Here, whole blood was collected in an 8.5 mL BD Vacutainer SSTII Advance Plus Blood Collection Tube during venipuncture. The tubes were processed within 1 h after collection by centrifugation for 10 min at 2,683 g at 20 °C. Four patient serum pools (HUM-P) used to study the interference effects and matrix effects were made by combining residual serum samples of different patients in such a way that individual patients were only used for a single pool. Serum was stored in aliquots at −80 °C before analysis.

Quantification of human albumin, immunoglobulin, total protein and CYFRA 21-1

For the quantification of albumin and total protein, colorimetric assays were used. Immunoglobulin (IgG) was quantified using an immunoturbidimetric assay. A commercially available ECLIA was used for the comparative quantification of CYFRA 21-1 (Roche Diagnostics, Rotkreutz, Switzerland). All assays were validated and performed on a Cobas platform (e801, Roche Diagnostics). The endogenous CYFRA 21-1 concentration in HUM-S was determined to be 1.3 ng/mL and the concentrations in HUM-P were 2.5, 3.4, 1.3 and 8.7 ng/mL.

Matrix effects and comparative study

Matrix effects were studied by spiking 100 ng/mL CYFRA 21-1 in six independent serum pools with initial CYFRA 21-1 concentrations ranging from 0.80 to 2.7 ng/mL. The calibration formula y=(3.0x + 0.62) · 10⁻², obtained after linear least-squares regression, was used to calculate the measured values. The differences between the obtained values and the nominal concentrations were calculated and expressed as a percent deviation (% bias). The CYFRA 21-1 concentrations in serum of 60 randomly selected study participants (20 benign disease, 20 stage I-II NSCLC and 20 stage III-IV NSCLC) were measured using both IA LC-MS/MS and ECLIA and compared using a Bland-Altman plot to determine the analytical bias and limit of agreement (LoA) between the methods. Receiver operating characteristics (ROC) curves were constructed to determine the area under the curve (AUC).

Signature peptide selection

The tryptic CYFRA 21-1 peptides were evaluated based on their uniqueness in the human genome, peptide length, presence of the unstable amino acids and detectability after IA QTOF LC-MS^e evaluation (Figure 1, Supplementary Figure 1). Peptide AALEDTLAETEAR (amino acids 318–330) met all criteria and was selected as SP and AALEDTLAETEA[R_¹³C₆, ¹⁵N₄] was designed as IS. Optimal multiple reaction monitoring (MRM) transitions for both peptides were found to be 695.3>676.2 (y6 fragment, quantifier) and 695.3>1,005.4 (y9 fragment, qualifier) for the SP and 700.3>686.2 for the IS (Supplementary Table 1). The qualifier:quantifier ratio was used for quality control.

Evaluation of interference effects

During IA purification, an interference effect can be caused by non-specifically bound substances that hinder the binding of the target analyte to the antibody [27]. To elucidate the presence of potential interference effects, 100 ng/mL CYFRA 21-1 was spiked into four different matrices; PBS with 5 % bovine serum albumin (BSA), BOV containing endogenous bovine CYFRA 21-1 which lacks the human SP sequence, purchased male AB serum (HUM-S) and four independent patient serum pools (HUM-P). IA LC-MS/MS analysis of these samples revealed large differences between the CYFRA 21-1 signals, expressed as the peak area ratio of the SP and IS, in the different matrices (Figure 2A). The maximum signal was obtained when using the least complex matrix (BSA), while the signal decreased by almost half upon the use of the purchased sera (BOV and HUM-S) and additionally halved in case of pooled patient serum (HUM-P). To confirm that the isolation efficiency decreased upon increased matrix complexity, the initial CYFRA 21-1 concentration spiked in the different matrices and the remaining CYFRA 21-1 concentration in the supernatant after IA purification was measured using ECLIA and used to calculate the percentage of uncaptured CYFRA 21-1. As shown in Figure 2B, a larger mean percentage of uncaptured CYFRA 21-1 remained in the supernatant of commercial serum (22 and 24 % for BOV and HUM-S respectively) compared to BSA (14 %). After isolation in patient serum, the highest percentage of CYFRA 21-1 remained in the supernatant (38 %). These results indicate the presence of significant interference effects during IA purification upon increased matrix complexity. No interference effects were seen during ECLIA (<20 %), which is expected to be a result of the low serum volume and large dilution factor used in this assay.

Figure 2:

Interference effects. (A) CYFRA 21-1 isolation in four different matrices revealed a decreased signal (mean + individual datapoints) upon increased matrix complexity. (B) The percentage of uncaptured CYFRA 21-1 increased in more complex biological matrices. (C) Reduced albumin, IgG and total protein concentrations were detected in HUM-S compared to HUM-P. (D) IgG (cetuximab) induced large interference effects during CYFRA 21-1 isolation. CYFRA 21-1, cytokeratin 19 fragment 21-1; BSA, PBS with 5 % bovine serum albumin; BOV, bovine serum; HUM-S, purchased male AB serum; HUM-P, four independent patient serum pools; IgG, immunoglobulin.

Common interferents are high abundant proteins including albumin and IgGs, which represent 60–90 % of the total serum protein concentration [28, 29]. As shown in Figure 2C, the albumin, IgG and total protein concentrations in HUM-S were indeed substantially lower compared to HUM-P (BOV could not be measured as the assays were compatible with human serum solely).

As the CYFRA 21-1 signal in BSA was substantially higher, albumin was not expected to cause the large interference effect in the serum matrices. To study the role of IgG, a monoclonal IgG1 antibody (cetuximab) dissolved in PBS was titrated in 100 ng/mL CYFRA 21-1, resulting in a large decrease in CYFRA 21-1 signal upon the presence of IgG (Figure 2D). This shows that IgG is significantly influencing the capture efficiency, which partially explains the differences between the studied matrices. The remaining interference effects, i.e. the difference between HUM-S and HUM-P, could not be assigned to specific proteins.

Protein precipitation

Multiple strategies were evaluated to decrease interference effects, such as ultrafiltration, IgG denaturation by disulfide reduction and several precipitation methods (data not shown). Only AS precipitation, which is a commonly used approach to salt out proteins [30], was compatible with the IA LC-MS/MS assay and successfully reduced the interference effects. Here, HUM-S was spiked with 100 ng/mL CYFRA 21-1, precipitated using increased v/v percentages of AS, the pellet was dissolved in PBS and the CYFRA 21-1, albumin, IgG and the total protein concentrations were measured using commercially available assays. Optimal CYFRA 21-1 precipitation was achieved when using 50 % v/v AS, at which 9.1 % of the total albumin fraction, nearly all IgG and around 33 % of the total protein fraction was precipitated, which is in agreement with previous findings of high abundant protein precipitation (Figure 3A–D) [30, 31]. Since the IgG depletion pattern was similar to the one of CYFRA 21-1, AS precipitation could not solve all interference effects, e.g. caused by IgG. However, incorporation of AS precipitation in the IA LC-MS/MS assay significantly improved the absolute CYFRA 21-1 signal in HUM-P towards the same level as BOV (Figure 3E). This allowed the use of BOV as a surrogate matrix for calibration purposes, which is beneficial compared to human serum as it does not contain endogenous human CYFRA 21-1.

Figure 3:

AS precipitation. (A) AS precipitation pattern of CYFRA 21-1, (B) IgG, (C) albumin and (D) total protein. (E) AS precipitation followed by IA purification and LC-MS/MS analysis in BOV and HUM-P revealing no significant differences between the matrices (n=4). AS, ammonium sulfate; CYFRA 21-1, cytokeratin 19 fragment 21-1; IgG, immunoglobulin; IA, immunoaffinity; LC-MS/MS, liquid chromatography-tandem mass spectrometry; BOV, bovine serum; HUM-P, four independent patient serum pools.

Assay calibration and validation

A calibration curve was analyzed on each of four subsequent days using weighted linear least-squares regression (1/x²). Linearity was achieved within the range of 1.0–100 ng/mL CYFRA 21-1 (R²=0.98) (Figure 4A). The LLoQ was established at 1.0 ng/mL (Figure 4B). No carry-over was observed and blank BOV samples did not show a detectable peak at the retention time of the SP nor the IS thus ensuring high selectivity.

Figure 4:

Analytical validation. (A) Calibration curve of CYFRA 21-1 (4 days). (B) LC-MS/MS quantifier signal at LLoQ (green, 1.0 ng/mL) overlaid with four blank BOV samples (gray). (C) Bland-Altman plot and (D) boxplots of the CYFRA 21-1 concentration in benign (n=20), stage I/II NSCLC (n=20) and stage III–IV NSCLC (=20) patients measured using ECLIA and LC-MS/MS. %Difference=100((ECLIA – LC-MS/MS)/average). CYFRA 21-1, cytokeratin 19 fragment 21-1; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LLoQ, lower limit of quantification; BOV, bovine serum; ECLIA, electrochemiluminescence assays; IA, immunoaffinity; LoA, limit of agreement.

The within run and between run precision and overall accuracy were determined in 3 days, for which at each day 4 QC levels were analyzed in fivefold (Table 1). All precision and accuracy values were <15 %, which is well within the acceptance criteria of the EMA guidelines (<15 % for all QC levels except samples at the LLoQ, which should not exceed 20 %). At low QC level, the calculated between run mean squares was smaller than the within run mean squares, resulting in a between run precision of 0 %.

Matrix effects

LC-MS is known to suffer from matrix effects during ionization as a result of co-elution of other substances that can cause signal suppression or enhancement [32], [33], [34]. To this end, serum of six healthy individuals was spiked with 100 ng/mL CYFRA 21-1 and the calibration curve was used to determine the measured values. As shown in Table 2, all measured values were within tolerance criteria (<20 %). Of note, the detected values were slightly lower than the nominal concentration.

Comparative study

Samples of 20 patients with benign lung diseases, 20 stage I-II NSCLC patients and 20 stage III-IV NSCLC patients were analyzed with the developed CYFRA 21-1 LC-MS/MS assay and compared to a commercially available ECLIA. By combining the analytical results, with exclusion of samples below the LLoQ of the LC-MS/MS assay (n=11), the analytical bias between the methods was determined to be −3.3 % (LoA −75 and 69 %), as shown in the Bland-Altman plot (Figure 4C). The concentrations in sera of patients with benign diseases, stage I-II NSCLC and stage III-IV NSCLC were comparable for both assays, where samples below the LLoQ of the LC-MS/MS assay were assigned as 1.0 ng/mL (Figure 4D). The AUC of the ROC curves to discriminate benign diseases from NSCLC was 0.79 ± 0.057 for LC-MS/MS and 0.83 ± 0.052 for ECLIA (Supplementary Figure 2). However, the limited number of study samples and the 11 samples that were below LLoQ of the LC-MS/MS assay do not allow to draw significant conclusions on the diagnostic value of the assay.