Detection of serum M-protein in acetonitrile precipitates by MALDI-TOF mass spectrometry: A novel,low-cost methodology

Abstract

Background

Several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis. We report the results of a novel, low-cost, reagent-based extraction process using acetonitrile (ACN) precipitation to enrich for κ and λ light chains which can be analysed by MALDI-TOF MS.

Methods

Institutional Ethics committee approval was obtained. Serum samples from patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, AL amyloidosis and Waldenström macroglobulinemia (WM) underwent ACN precipitation. The images obtained were overlaid on apparently healthy donor serum samples to confirm the presence of M-protein. A sample was considered positive for M-protein if there was a sharp or broad peak within the κ or λ mass/charge (m/z) range: m/z- [M + 2H]²⁺: 11,550–12,300 Da and λ m/z- [M + 2H]²⁺: 11,100–11,500 Da. Images were acquired at a m/z range of 10,000–29,000 Da. Corresponding serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (IFE) and serum free light chain (sFLC) assay by nephelometry were performed for all the samples.

Results

Two-hundred-and-two serum samples were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). All the SPEP positive samples were identified by MALDI-TOF MS. Out of 179 samples positive for M-protein by IFE, MALDI-TOF MS was positive in 176 samples (98%). Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively.

Conclusions

This study demonstrates the feasibility of qualitatively identifying M-protein without the need for antibody-based immunoenrichment, making the technique cost-effective.

Keywords

Monoclonal protein identification light chains MALDI-TOF mass spectrometry

Introduction

Monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), plasmacytoma, light chain (AL) amyloidosis and Waldenström macroglobulinemia (WM) belong to a group of disorders known as clonal plasma cell dyscrasias.^1–4 These disorders are diagnosed by detecting an abnormally secreted monoclonal protein (M-protein) in the blood and sometimes in the urine. Around 2–3% have non-secretory MM, that is, absence of M-protein.¹

M-protein is analysed by a multitude of tests, including serum/urine protein electrophoresis (SPEP/UPEP), serum/urine immunofixation (IFE) and serum/urine free light chains (sFLC/uFLC). The combination of these tests can be laborious, time-consuming and expensive.⁵

Recent studies have demonstrated the feasibility of employing matrix-assisted laser desorption ionization mass spectrometry-time of flight mass spectrometry (MALDI-TOF MS) to detect M-protein by analysing immunoglobulin isoforms and light chains using isotype-specific nanobodies in MM and AL amyloidosis termed MASS-FIX.^6–10 MASS-FIX has demonstrated superior analytical sensitivity to IFE.¹¹ MASS-SCREEN is another rapid and accurate screening technique using $κ$ and $λ$ nanobodies to look for the presence of M-protein.⁷

The advantage of using MALDI-TOF MS analysis is based on the ability to measure the mass to charge (m/z) ratios of the light chains: $κ$ m/z ([M + 2H]²⁺: 11,550–12,300 Da, [M + H]⁺: 23,100–24,600 Da), and $λ$ m/z ([M + 2H]²⁺: 11,100–11,500 Da, [M + H]⁺: 22,200–23,100 Da) and high throughput automated analysis.⁷ In a healthy individual, the secretion of immunoglobulins by plasma cells in the bone marrow is polyclonal, with many plasma cells secreting these proteins. Upon the analysis in a mass spectrometer, the polyclonal nature of the secretion would give rise to a spectrum indicating a Gaussian distribution pattern with no single protein clone ( $κ$ or $λ$ ) being predominant.¹² However, due to the increased secretion of antibodies by a single clone in malignancy, there is a distinct secretion pattern contrary to the Gaussian distribution pattern and is reflected in the MALDI-TOF MS analysis. This distinction in the protein profile pattern between the healthy and diseased state is diagnostic for monoclonal gammopathy. Another necessary consequence of the study of antibody light chains using MALDI-TOF MS is that the m/z measurements obtained from the instrument can help determine the light chain involvement, which can be categorized as either $κ$ or $λ$ .

Another unique advantage of MALDI-TOF is its use for disease response assessment in patients with IgG $κ$ MM who are on therapeutic monoclonal antibodies belonging to the same class-IgG $κ$ subgroup, for example, daratumumab (anti-CD38 monoclonal antibody).^11,13,14 Among the most clinically relevant applications is the MRD (minimal or measurable residual disease) assessment in the peripheral blood by MALDI-TOF MS, which has the potential to reduce the frequency of bone marrow examinations for MRD assessment at different time points.¹⁵

Here we report the feasibility of an alternate low-cost, reagent-based extraction process that can enrich the light chains- $κ$ and $λ$ proteins and is simultaneously compatible with MALDI-TOF MS.

Methods

Samples

Control and patient samples were collected after obtaining Institutional Ethics Committee approval for carrying out a study on biomarker discovery in plasma cell disorders. The work was carried out in accordance with the Declaration of Helsinki after obtaining written informed consent from patients. Excess control serum samples were obtained from healthy blood product donors who provided a sample for preliminary screening. Patients with confirmed plasma cell disorders who underwent SPEP, serum IFE and sFLC were included for MALDI-TOF MS analysis using the extra serum sample. Descriptive statistics were carried out using IBM SPSS Statistics v28.0.1.0 (142). The chi-square test was used to analyse the statistical significance of concordance between various tests.

Serum protein electrophoresis, serum IFE and sFLC analysis

Serum protein electrophoresis was performed on the Minicap/FP fully automated capillary electrophoresis analyser (Sebia, France), and serum total protein concentration was determined by the colourimetry method using biuret reagent on the BA400 fully automated clinical chemistry analyser (manufactured by Biosystem S.A., Spain). From the total protein value, the software automatically quantified each fraction (𝛼1, 𝛼2, β1, β2 and γ globulins). With the help of a pointer on the screen, the M-protein area was marked manually. The instrument automatically calculated the concentration of M-protein from the total protein value.

Serum IFE was performed on SAS-3 9IF gel (Helena Biosciences) or the Hydrasys 2 scan focusing gel electrophoresis (Sebia, France) instrument and reported.

Serum free light chain was analysed on BN II Nephelometer by Siemens using the Binding Site kit. IgD and IgE immunotyping was not performed. Based on the sFLC readings, a sample was labelled as $κ$ or λ monoclonal gammopathy if the sFLC ratio was >1.65 or <0.26, respectively. Serum IFE and sFLC analyses were sent to independent laboratories for external validation of the MALDI-TOF MS results.

Reagent-based extraction

The serum fraction was separated from whole blood by centrifugation at 5000 rpm for 15 min and stored at −80°C until further analysis. Twenty-five $μ L$ of the serum was taken in a fresh 1.5 mL centrifuge tube and mixed with 400 $μ L$ of extraction solution-50% acetonitrile (ACN) prepared in LC-MS grade water, allowing the protein fraction to precipitate. Mass spectrometry grade ACN and LC-MS grade water were both purchased from Sigma-Aldrich. The mixture was incubated at room temperature (28°C) for 5 min, centrifuged at 12,000 rpm for 10 min and the protein precipitate was collected. The protein precipitate was mixed with equal volumes, that is, 400 $μ L$ of 20% ACN (prepared in LC-MS grade water) and washed. The solution was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and the precipitate was retained. Then, the precipitate was reconstituted in 200 $μ L$ reconstitution buffer: 10% formic acid (FA) plus 50 mmol/L tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and centrifuged at 12,000 rpm for 10 min. Tris(2-carboxyethyl) phosphine hydrochloride was purchased from Tokyo Chemical Industry Co., LTD, and formic acid with 98% purity was purchased from Sigma-Aldrich.

Western blot analysis of serum ACN precipitates

Acetonitrile precipitation was performed on all the serum samples as described above. The ACN precipitates were subjected to SDS PAGE electrophoresis using 12% SDS PAGE gel. Following western blot transfer onto a nitrocellulose membrane, the blot was incubated in a blocking solution (3% non-fat dry milk in TBST buffer) for 1 h. Next, the blot was incubated in primary antibody solution of either biotin-labelled mouse $κ$ or $λ$ antibody at a dilution of 1:1000 in blocking buffer overnight at 4°C. Next, the blot was washed three times for 10 min each in TBST wash buffer. Next, the blot was incubated in a solution consisting of HRP-streptavidin at a dilution of 1:10,000 in TBST for 1 h at room temperature. The blot was washed three times for 10 min each in TBST and incubated in DAB (diaminobenzidine) solution prepared by dissolving 0.1 gm in 10 mL of distilled water. The blot was developed till a brown-coloured precipitate formed in the positive control. The reaction was stopped by washing the blot in distilled water.

Antibody immunoenrichment

The procedure for immunoenrichment was performed as previously described⁷ with some modifications. Due to the non-availability of agarose beads and nanobodies targeting $κ$ and $λ$ constant region domains, our protocol was modified to include direct use of biotin-labelled $κ$ and $λ$ antibodies and subsequently immobilized in streptavidin magnetic beads. Twenty-five $μ L$ of the serum was diluted in 225 $μ L$ of LC-MS grade water and added to a mixture of 5 $μ L$ each of either $κ$ or $λ$ antibody immobilized in streptavidin beads. Captureselect Biot A-light chain $κ$ HU and light chain $λ$ HU antibodies, and MyOne Streptavidin T1 beads were purchased from Thermo Fisher Scientific. The diluted serum and the antibody-coated bead mixture were incubated at room temperature for 45 min. This mixture was then washed five times with LC-MS grade water and eluted with 40 $μ L$ elution buffer (10% FA+50 mmol/L TCEP). This was subsequently vortexed for 15 min.

MALDI-TOF analysis

One $μ L$ of the protein solution was spotted along with the MALDI matrix (alpha-cyano-4-hydroxycinnamic acid + 10 g/L in 50% ACN + 0.1% TFA) onto a MALDI ground steel plate and allowed to dry. The samples were then analysed using Ultraflex^TM LT, Bruker MALDI/TOF-TOF mass spectrometer. Mass measurement was analysed with a summation of 500–2500 shots depending on the intensity of the monoclonal peak. The mass spectra for each sample were exported to FlexAnalysis 3.3 (Bruker Daltonics) and the background subtracted. Before analysis, the MALDI-TOF mass spectrometer was calibrated using a protein standard (Bruker Daltonics). The signal-to-noise threshold (S/N) for peak picking was set at 3, and the absorbance intensity threshold and relative intensity threshold were set at 5.

Interpretation of M-protein

A sample was considered positive for M-protein if there was a sharp or broad peak within the $κ$ or $λ$ m/z mass spectra range.⁷ All the patient samples were overlaid on the mass spectra from a healthy serum control sample with the polyclonal distribution of $κ$ and $λ$ light chains. A sample was labelled $κ$ or $λ$ depending on the m/z value of the M-protein that is, $κ$ m/z ([M + 2H]²⁺: 11,550–12,300 Da, [M + H]⁺: 23,100–24,600 Da), and $λ$ m/z ([M + 2H]²⁺: 11,100–11,500 Da, [M + H]⁺: 22,200–23,100 Da).⁷

Results

Identification of $κ$ and $λ$ light chains in ACN precipitates of serum

κ and λ light chain M-protein standards were spotted at different concentrations: 3–12 ng/µL for $κ$ and 2–20 ng/µL for λ. The peak profile was assessed using the above-described MALDI-TOF data acquisition parameters. In our analysis, the minimum concentration at which we could observe [M + 2H]²⁺ peak profiles for the κ and λ standards in 0.1% TFA was determined at 3.7 ng/µL for κ and 2.8 ng/µL for λ. Next, we tested a range of concentrations for ACN from 10% to 100% to induce precipitation of a fixed volume of serum (20 μL); it was found that using 50% ACN, the precipitate obtained was optimal for analysis (data not shown). We spotted a range of sample (reconstituted ACN precipitate) dilutions on the MALDI plate: neat, 1:10, 1:25, 1:50 and 1:100. Neat and 1:10 dilution were found to generate spectrum profiles distinct from the background signal (data not shown). The neat sample was spotted on the MALDI plate for all control and patient samples. All the images were acquired at an m/z range of 10,000–29,000 Da to include the [M + H]⁺ and [M + 2H]²⁺ charge states for κ and λ (Figure 1(a) and (b)). [M + 2H]²⁺ charge state images were chosen for further analysis due to superior visual sensitivity, as already described in the MASS-SCREEN study.⁷ Further, the ACN precipitates from two serum samples, one with κ M-protein and the other with λ M-protein, were subjected to western blot analysis to confirm the presence of light chains and results indicated that the ACN precipitates contained the κ and λ M-protein. The amount of serum sample required for analysis was tested in the range from 10 to 100 $μ L$ . 25 $μ L$ was chosen for further analysis because at this loading amount, the intensity of the monoclonal peak made the detection unambiguous at the set S/N, absorbance intensity threshold and relative intensity threshold, with no interfering peaks within the κ and λ m/z range.

Figure 1.

(A) FLC mass spectra with [M + H]⁺ and [M + 2H]⁺² charge states: Serum with $λ$ monoclonal gammopathy by acetonitrile precipitation. [M + H]⁺- 22,856 Da and [M + 2H]⁺- 11,428 Da. (insert: Western blot confirmation of $λ$ light chain present in the ACN precipitate). (B) FLC mass spectra with [M + H]⁺ and [M + 2H]⁺² charge states: Serum with $κ$ monoclonal gammopathy by acetonitrile precipitation. [M + H]⁺- 24,274 Da and [M + 2H]⁺- 12,137 Da. (insert: Western blot confirmation of $κ$ light chain present in the ACN precipitate).

Validation of ACN precipitation results with immunoenrichment with $κ$ and $λ$ antibodies

Immunoenrichment was performed for two $κ$ M-protein samples and two $λ$ M-protein samples. The mass spectra were identical, with the light chain m/z falling within the range reported by ACN precipitation (Figure 2(a) and (b) and Figure 3(a) and (b)).

Figure 2.

Serum sample with $κ$ monoclonal gammopathy. (A) Light chain mass spectra showing a sharp $κ$ M-peak overlaid on a healthy serum control by acetonitrile precipitation. (B) Immunoenrichment with $κ$ antibody tagged to streptavidin beads from the same patient showing a sharp M-peak overlaid over a healthy serum control and streptavidin bead control with no peak.

Figure 3.

Serum with $λ$ monoclonal gammopathy. (A) Light chain mass spectra showing a sharp M-peak overlaid on a healthy serum control by acetonitrile precipitation. (B) Immunoenrichment with $λ$ antibody tagged to streptavidin beads from the same patient showing a sharp M-peak overlaid on a healthy serum control and streptavidin bead control with no peak.

Acetonitrile precipitation of the control serum samples

The Gaussian distribution of light chains was obtained by analysing 20 control samples from the extra serum of apparently healthy blood donors. All the control samples underwent SPEP. Serum total protein was within normal limits for all samples, and SPEP did not demonstrate an M-protein (Figure 4).

Figure 4.

Mass spectra at [M + 2H]²⁺ charge state of all the healthy serum controls (N = 20). Gaussian distribution of polyclonal $κ$ and $λ$ light chains: $κ$ m/z ([M + 2H]²⁺: 11,550–12,300 Da and $λ$ m/z ([M + 2H]²⁺: 11,100–11,500 Da.⁷

Patient samples

Two-hundred-and-two serum samples from patients with plasma cell disorders were included in the study: MM- 184 (91%); AL amyloidosis- 2 (1%); plasmacytoma- 8 (4%); MGUS- 6 (3%) and WM- 2 (1%). One-hundred-and-nineteen samples (59%) were taken from patients with newly diagnosed plasma cell disorders who received no treatment. All the others were either on or completed treatment.

All the samples that were positive for M-protein by SPEP were identified by MALDI-TOF MS. However, out of 179 samples that were positive for M-protein by IFE, MALDI-TOF MS identified an M-protein in 176 samples (98%). Of these three samples identified by IFE and not by MALDI-TOF MS, one sample had a normal sFLC ratio. The other two samples that were negative by MALDI-TOF MS were identified as IgG $κ$ by IFE. Among samples that were SPEP negative/IFE positive (N = 55), MALDI-TOF MS identified 52 samples (95%). This demonstrates the ability of MALDI-TOF MS to correlate well with IFE. Among samples that were SPEP and IFE negative (N = 26), MALDI-TOF MS identified an M-protein in 11 samples (42%). Of these 11 samples, six samples had an abnormal sFLC ratio as well. Among the remaining five samples: 2- subsequent disease relapse, 1- no follow-up sample available, 1- subsequent IFE showed a thin discrete monoclonal band corresponding to IgG $κ$ and 1- subsequent MALDI-TOF MS was negative.

Compared to IFE, the sensitivity and specificity of M-protein identification by MALDI-TOF MS were 98.3% and 52.2%, respectively. With IFE as the reference, the presence or absence of M-protein by MALDI-TOF MS was concordant in 191 out of 202 samples (95%, p < 0.001)

With a routine serum panel (SPEP, IFE and sFLC) as the reference, the presence or absence of M-protein by MALDI-TOF MS was also concordant in 191 out of 202 samples (95%, p < 0.001). (Table 1) In the entire cohort, MALDI-TOF MS did not identify an M-protein in 15 samples. Of these, four samples had abnormal sFLC ratios above the reference range (0.26–1.65); the abnormal sFLC ratios ranged from 2.29–10.2. M-protein was absent by SPEP in all the 15 samples. This demonstrates the importance of continuing sFLC testing to identify a monoclonal protein.

Table 1.

Comparison between SPEP, IFE, sFLC and MALDI-TOF MS.

	MALDI-TOF MS+	MALDI-TOF MS-
SPEP+/IFE+	124 (100%)	0
SPEP-/IFE+	52 (95%)	3 (5%)
SPEP-/IFE-	11 (48%)	12 (52%)
IFE+/sFLC+	135 (99%)	1 (1%)
IFE-/sFLC+	6 (67%)	3 (33%)
IFE+/sFLC-	41 (95%)	2 (5%)
IFE-/sFLC-	5 (36%)	9 (64%)

Six serum samples had characteristic polytypic peaks with a significant rightward shift (beyond the normal $κ$ range) in the mass spectra. (Figure 5) This pattern has been described previously resulting from the post-translational modification of light chains, usually glycosylation. The characteristics of these light chain spectra have been previously described.¹⁴ As reported in the literature, these glycosylated light chains can reduce the ionizing efficiency of the primarily involved monoclonal light chain (Figure 5).

Figure 5.

Mass spectra at [M + 2H]²⁺ charge state demonstrating the characteristic polytypic peaks suggestive of a glycosylated light chain (m/z: 12,569 Da).

The concordance between IFE (as reference) and MALDI-TOF MS for the correct identification of

κ

M-protein was 92% (97/105); four samples showed an M-protein in the λ range (m/z values: 11,252 Da, 11,253 Da, 11,481 Da and 11,504 Da). (Table 2) The concordance between IFE (as reference) and MALDI-TOF MS for the correct identification of

λ

M-protein was 84% (62/74); 11 samples showed an M-protein in the

κ

m/z range: 11,454–11,650 Da.

Table 2.

MALDI-TOF MS agreement with IFE.

IFE	N	MALDI-TOF MS+	MALDI-TOF MS-	Agreement, %
IgG	116	113	3	97
IgA	34	34	0	100
IgM	2	2	0	100
FLC	27	27	0	100
Negative	23	11	12	48

All the lab members who participated as mass spectra analysts were given a training set and static images of the mass spectra. The mass spectra were assessed by three blinded analysts (authors G. Gopisetty, S. Jayavelu and S. Dhanasekar). N. Mehra was the unblinded analyst. A final consensus was obtained from all four analysts (agreement meant consensus obtained among at least three out of four analysts). Consensus was achieved in 96% (193/202) of the serum samples. Among the samples where consensus could not be achieved, the most common reason was low signal intensity, and the other reason was possible albumin contamination as described previously.⁷ The images were zoomed in on the X-axis for better clarity for the samples with low signal intensity. After including one more wash step with 20% ACN, the contamination peaks were no longer visible, making the M-protein call unambiguous. There was complete consensus after the additional nine images were either zoomed in or after including the extra wash step.

MALDI-TOF MS analysis of samples with AL amyloidosis

In this report, two patients had AL amyloidosis confirmed by Congo red staining of the tissue biopsy. There was discordance in the monoclonal light chain identified by MALDI-TOF MS versus serum IFE and sFLC in both patient samples. The serum IFE and sFLC were suggestive of $λ$ light chain involvement. However, MALDI-TOF MS revealed an M-protein within the $κ$ range, (m/z values: 11,555 Da and 11,573 Da). This phenomenon of a shift in the light chain spectra to the right, that is, ‘heavy’ molecular mass in renal AL amyloidosis has been previously described.^10,16,17

Discussion

This study demonstrates the feasibility of qualitatively identifying M-protein without requiring immunoglobulin light chain-specific antibody-based immunoenrichment using agarose or sepharose beads.¹² Since antibodies are not required for M-protein analysis by MALDI-TOF MS using this method, a lack of specificity would be a possible drawback of the present methodology. However, immunoblot analysis of the ACN precipitates has revealed the presence of immunoglobulin light chains indicating that the ACN precipitation of serum resulted in enrichment of immunoglobulin light chains.

Using IFE as the gold standard, the sensitivity of M-protein identification by MALDI-TOF MS was 98%. However, the specificity was only 52%.

Our results are in line with studies published previously regarding the excellent analytical sensitivity of M-protein detection by MALDI-TOF MS.^6–8 One disadvantage of this technique is that the call between $κ$ and $λ$ was made by identifying the monoclonal peak within the mass spectra range as pre-defined. For $κ$ M-protein, the concordance between IFE and MALDI-TOF MS was 92% and for $λ$ , it was 84%. The light chain could not be accurately identified in the cases where a rightward shift was observed by MALDI-TOF MS either, which could be due to possible glycosylation of LCs or when the m/z value fell in a region of overlap in the $κ$ and $λ$ m/z ranges mostly observed between m/z ∼11,450 Da and 11,650 Da. All except two samples positive for $κ$ M-protein identified by IFE but showing a $λ$ M-protein by MALDI-TOF MS and vice versa had m/z values falling within this overlapping range.

In the present report, the analysis of M-protein by ACN precipitation technique was qualitative and was defined by the m/z value of the aberrant light chain clone. Further studies will focus on developing this methodology to analyse immunoglobulin isoforms and quantitative determination of M-protein, as already reported with nanobody enrichment called MASS-FIX.⁶ MASS-FIX was found to be superior to IFE in predicting progression-free survival and overall survival outcomes.¹⁸ We envisage that ACN precipitation methodology can also serve as a useful screening technique for M-protein analysis by MALDI-TOF MS in a haematology lab in place of SPEP and serum IFE. In the future, given the cost-effectiveness of the ACN precipitation methodology relative to the antibody-based M-protein enrichment by MALDI-TOF MS, studies will be undertaken to identify the predictive value of this test compared to other peripheral blood and bone marrow response assessment tools. This would be particularly relevant in low-income and low-and-middle-income countries where the out-of-pocket expenses for cancer diagnostics and treatment are high due to limited health insurance coverage.¹⁹

To conclude, we have developed a low-cost, reagent-based extraction process using ACN precipitation to enrich $κ$ and $λ,$ for the screening and qualitative analysis of M-protein.

Footnotes

Acknowledgements

We thank the Medical Oncology trainees and Ms Roja from the Blood Bank at the Institute for helping with sample collection. Presented at the American Society of Hematology (ASH) 2022 62nd Annual Meeting, Virtual, December 5-8, 2020 (poster) citation: Nikita Mehra, Gopal Gopisetty, Jayavelu S, et al: Detection of M-protein in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry: A Novel Methodology. Blood 136:36-37, 2020 (Suppl 1; abstr session 803); the International Myeloma Society Annual Meeting, Vienna, Austria, September 8-11, 2021 (poster) citation: Nikita Mehra, Gopal Gopisetty, Jayavelu S, et al: Detection of M-protein in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry: A Novel Methodology. Clinical Lymphoma, Myeloma and Leukemia 21, 2021(suppl 2, S94; abstr #101). At the conference, N. Mehra received the young investigator award for exemplary abstract for the work. International Myeloma Society Annual Meeting, Los Angeles, CA, August 25-27, 2022 (poster): Nikita Mehra, Gopal Gopisetty, Jayavelu S, et al: Screening Technique for M-proteins in Plasma cell Disorders in Acetonitrile Precipitates of Serum using MALDI-TOF Mass Spectrometry: A Comparison with Serum Immunofixation Electrophoresis and Serum Free Light Chain Assay. The published work is a part of Nikita J Mehra’s Ph.D. Thesis of The Tamil Nadu Dr. M.G.R. Medical University.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: GG, NM, SJ, Indian provisional patent application no: 202041009443, filed on 5 March 2020.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by funds from the Cancer Institute (WIA).

Ethical approval

Control and patient samples were collected after obtaining Institutional Ethics Committee approval for carrying out a study on biomarker discovery in plasma cell disorders. Cancer Institute (WIA)- Institutional Ethics Committee, Reference Number: IEC/2019/13.

Guarantor

GG.

Contributorship

NM- Conceptualisation; methodology; data curation; investigation; resources; validation; formal analysis; writing- original draft, review and editing; visualisation; project administration.

GG- Conceptualisation; methodology; software; validation; formal analysis; investigation; resources; data curation; writing- original draft, review and editing; visualisation; project administration.

SJ and SD- Software; validation; investigation; data curation; formal analysis; visualisation.

RA- Conceptualisation; methodology; validation; resources; writing- original draft, review and editing.

JPK, PK and KK- Investigation; resources; writing- review and editing.

SR- Formal analysis; resources; writing- original draft, review and editing; visualisation.

TR- Conceptualisation; methodology; formal analysis; resources; writing- review and editing; visualisation; supervision; funding acquisition; project administration.

Correction (July 2024):

Article updated online to include ‘The published work is a part of Nikita J Mehra’s Ph.D. Thesis of The Tamil Nadu Dr. M.G.R. Medical University.’ in the ‘Acknowledgements’ section.

ORCID iDs

Nikita Mehra

Gopal Gopisetty

Jayachandran Perumal Kalaiyarasi

Parathan Karunakaran

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Detection of serum M-protein in acetonitrile precipitates by MALDI-TOF mass spectrometry: A novel,low-cost methodology

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Methods

Samples

Serum protein electrophoresis, serum IFE and sFLC analysis

Reagent-based extraction

Western blot analysis of serum ACN precipitates

Antibody immunoenrichment

MALDI-TOF analysis

Interpretation of M-protein

Results

Identification of κ and λ light chains in ACN precipitates of serum

Validation of ACN precipitation results with immunoenrichment with κ and λ antibodies

Acetonitrile precipitation of the control serum samples

Patient samples

MALDI-TOF MS analysis of samples with AL amyloidosis

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

Correction (July 2024):

ORCID iDs

References

Identification of $κ$ and $λ$ light chains in ACN precipitates of serum

Validation of ACN precipitation results with immunoenrichment with $κ$ and $λ$ antibodies