June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Research Paper

Application of MALDI-TOF MS analysis for rapid discriminationof free immunoglobulin light chains, kappa and lambda isotypes
in patients with monoclonal immunoglobulin gammopathy
1

1

2

3

Shuichi Asano , Ryota Masutani , Kazuhito Ueda , Kazuo Igarashi ,Takashi Nirasawa , Meri Kubota , Yoko Tabushi-Matsumura ,
4

1

1

1
1, 5
Naofumi Osaka , Toyofumi Nakanishi *1

Central Clinical Laboratory, Osaka Medical and Pharmaceutical University Hospital,
2‒7 Daigaku-cho, Takatsuki, Osaka 569‒8686, Japan2
Department of Clinical Laboratory, Faculty of Health Science, Kansai University of Health Sciences,2‒11‒1 Wakaba, Kumatoricho, Sennan-gun, Osaka 590‒0482, Japan3
Department of Forensic Medicine, Faculty of Medicine, Kinki University,
2‒377 Ohnohigashi, Osakasayama, 587‒8511, Japan
4
Bruker Daltonics KK, Division of Application, 1‒8‒29 Nishimiyahara, Yodogawa-ku, Osaka 532‒0004, Japan
5
Research and Development Center, Osaka Medical Pharmaceutical University,
4‒20‒1 Nasahara, Takatsuki, Osaka 569‒1094, Japan
Abstract Among immunoglobulin free light chains (FLCs), kappa and lambda isotypes exist mainly in monomeric anddimeric forms. Under pathological conditions, the levels of total FLCs as well as the ratios between mono- and dimeric FLCs
signiﬁcantly change in samples from patients with monoclonal immunoglobulin gammopathy (MMG). The MALDI-TOFMS
technique, which would be correspond to the electrophoresis of proteins under a high vacuum, can be used to easily andrapidly detect any target proteins in body ﬂuids without any speciﬁc antibodies. We compared the eﬀectiveness of MALDI-TOF MS and immunoelectrophoresis techniques to detect FLCs in MMG patients. Our preparations were only treatedTMwith desalts and concentration using the SpinFilter 3K

and Aﬃ-Gel

TM

Blue resin without any antibodies. The ratiosof the relative ion intensities between [ca 45 kDa] and [ca 23 kDa] by MALDI-TOF MS were 0.054＋/−0.05 (n＝26) of
the κ -type, and those of the λ -type were 0.572＋/−0.45 (n＝12), respectively. The ratios of [ca 45 kDa] / [ca 23 kDa] wereconsisted with those of dimers and monomers in patients samples by immunoelectrophoresis techniques. Conclusion: The
ratios of the relative ion intensity at m/z ca 23 kDa and ca 45 kDa of FLCs in patients urine may facilitate the rapid discrimination between BJP κ - and λ -types in MMG.
Key words: Bence‒Jones protein, MALDI-TOF MS, monoclonal immunoglobulin gammopathy, monomer‒dimmer ratios,
rapid discrimination* Corresponding author

Introduction

Toyofumi Nakanishi

In multiple myeloma (MM) and other monoclonal immu-

Research and Development Center, Osaka Medical andPharmaceutical University, 2‒7 Daigaku-cho, Takatsuki, Osaka

noglobulinemia, monoclonal plasma cells proliferate andproduce either κ - or λ -type light chains. There are increases

569‒8668, Japan
Tel: ＋81‒72‒683‒1221, Fax: ＋81‒72‒684‒6548

in total amounts of the κ - and/or λ -type, and the ratios
between κ - and λ -types are abnormal. In addition, sinceE-mail: nakanisi@osaka-med.ac.jp
Received: April 14, 2021.

about 40% more light chains are produced than heavy

Accepted: May 27, 2021.

chains, any the light chain that cannot bind to a heavy chain
is called a free light chain (FLC) and released extracellu-

Epub June 18, 2021.
DOI: 10.24508/mms.2021.06.007
47

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1‒3)

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Medical Mass Spectrometry Vol. 5 No. 1

these reasons, many kinds of spin column devices ﬁlledwith group-speciﬁc resins may be more suitable to rapidly.

Immunoglobulin FLCs, κ - and λ -types, exist mainly inmonomeric, dimeric, and multimeric forms

4,5). Under

separate the target proteins in body ﬂuids, such as urine,
plasma, and cerebral spinal ﬂuid, without speciﬁc antibod-

pathological conditions with an excessive production ofplasma cells, the levels of FLCs and ratios between mono-

ies against the targets.

and dimeric FLCs signiﬁcantly change. Especially, veryIn this study, we examined a total of 62 patients withhigh levels of FLCs were frequently reported in the urine ofmonoclonal immunoglobulin gammopathies, such as apatients with monoclonal immunoglobulin gammopathy
6‒8)(MMG), such as multiple myeloma (MM) . Various labo-

MM, including suspected cases to identify proteins proﬁles
by MALDI-TOF MS. For the simple and rapid preparation

ratory markers are used for the diagnosis and monitoring ofpatients with MM. Among protein components, monoclonalof patients urine, we applied SpinFilter 3K or followedTMwith Aﬃ-Gel Blue resin. The ratios of the ion intensities

peaks and/or BJPs are identiﬁable in virtually all patients
9‒12）. A major pitfall in the analysis of monowith the disease

between [ca 45 kDa] and [ca 23 kDa] may facilitate discrimination of κ - and/or λ -types of MMG.

TM

clonal immunoglobulin light chains in a MM patient s urine
is the unreliability of recognizing FLCs. Additionally, theconventional tests used for detecting Bence-Jones protein

Materials and Methods

are falsely negative in approximately one-half of patientssuspected of conﬁrmed MMG after receiving of informed

with MM. Because of the high urinary concentration ofconsent at Osaka Medical College Hospital. This study has

FLCs, spot urine has been used for the detection of FLCs inbeen approved by the Ethics Commission and the number isthe Clinical division - 969(2884). We selected 38 urine sam-Spot urines were collected from a total of 62 patients withpatients with suspected MM. Moreover, the amount of thelight chain excreted in the urine often correlates with disease progression. Generally, immunoassay techniques, suchples from those deﬁnitively diagnosed with MMG by immunoﬁxation and then analyzed them by MALDI-TOFMS. Aﬃ-

as immunoelectrophoresis, immunoﬁxation electrophoresis,Geland latex coagulating nephelometry, are routinely used for
13‒16). The immunoasthe screening of FLCs in body ﬂuids

and SpinFilter 3K

TM

Blue resin was purchased from Bio-Rad (Germany)TM

was from Merck (Germany).

say techniques essentially need speciﬁc FLC antibodies to

The standards of κ (P016) and λ (P017) chains of BenceJohn Protein (BJP) were purchased from Nordic-MUbio

detect FLCs in body ﬂuids, but false results frequently(Netherlands), and Sinapinic acid (P code: 101006397) for

might arise from the titer and speciﬁcity of the antibody

the ionization reagent was from Fluka (CA, USA). The
MALDI-TOF mass spectrometer was used at Autoﬂex

itself as well as some disadvantages of low reproduction,
17‒19).
limits of detection, and expert skills to detect FLCs
On the other hand, the MALDI-TOFMS technique,speed (Bruker Daltonics KK, Germany) and in a linear
mode at a mass range from 10,000 to 100,000.
TM
SDS-polyacrylamide gel, MultiGel II , and a runningso-called electrophoresis under a high vacuum, can be usedwith a wide range of molecular weights in body ﬂuids with

buﬀer were purchased from Cosmo Bio (Tokyo, Japan).
Monoclonal speciﬁc antibodies against κ (sc-52338) and. During the past threeλ (sc-52339) types of BJP were from Santa Cruz Biotech-decades, our group has applied the immunoprecipitation
technique to rapidly separate some disease-speciﬁc target

nology Inc. (CA, USA). Western blot (WB) of separated
proteins in the gels was performed with iBlots 2 Dry Blot-

molecules in patients sera, such as a carbohydrate-deﬁcienttransferrin, mutant transthyretin and superoxide dis-ting System

to easily and rapidly detect target proteins, such as FLCs,
20‒23)

or without speciﬁc antibodies

24‒26)(Thermo Fisher Scientiﬁc Inc. CA, USA)according to the manufacturer s manual. The other reagentswere from Nacalai Tesque (Kyoto, Japan). The quantiﬁca-

mutase-1 in patients sera with neurodegenerative diseases
prior to MS analysis

TM

TM. This immunoprecipitation istion analysis module of the Fusion FX7

software (ver.

more suitable to rapidly and simply purify target proteins in

17.01) was performed to calculate the ratio between [cabody ﬂuids than aﬃnity chromatography. However, inorder to more easily apply this MS technique to routine lab-

45 kDa] and [ca 23 kDa] of the Western blot (WB) analysis.

oratory tests, it might be very important to simply prepare

The patients urine used to diagnose MM was desaltedTMprior to MALand concentrated with SpinFilter 3K

target molecules from body ﬂuids prior to MS analysis. For

DI-TOF MS analysis. After this pretreatment, the analyte48

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

was diluted with distilled water 10 to 1000 times. For urine
showing severe proteinuria, we pretreated it with Aﬃ-

compared the ratios of the relative ion intensities between[ca 45 kDa] and [ca 23 kDa] of FLCs in MM patients by
MALDI-TOFMS could be used to rapidly discriminate

Blue resin to remove albumin. The 50‒500-times
diluted analyte was subjected to MALDI-TOF MS analysis.
TMGelbetween the κ - and λ -types of MM without any speciﬁc

The diluted analytes of MM patients and saturated sinapinicacid solution with 0.5% triﬂuoroacetic acid-50% acetoni-

antibodies against FLCs.

trile (w/w %: ﬁnal concentration) were equivalently added,
and then 1 μL of the mixture was dropped on the MAL-

in deﬁnite MM patients urines following immunoﬁxation
to compare MALDI-TOF MS analysis and gel electrophore-

DI-target, dried at room temperature, and then applied to
MALDI-TOF MS analysis in the linear mode. We checked

sis under non-reduction/WB/ECL detection. The protein

the m/z calibration with the protein I and II calibrator(Bruker Daltonics KK., Tokyo) before daily MS analysis.

at m/z 23297 and 24034, shown in two typical cases of the
BJP κ -type, and an ion at m/z 48224, shown in one case

Results and Discussion(Fig. 1a and 1b). On the other hand, the two typical cases of
the BJP λ -type clearly showed ions at m/z 22431 andFig. 1.

shows four typical cases of BJP κ - and λ -types

proﬁles of MALDI mass spectra were predominantly ions

In the present study, rapid and simple detection of FLCs
in spot urines derived from patients with monoclonal immu-

22823, with ions at m/z 44920 and 45731, respectively (Fig.
1c and 1d). The ratios of the ion intensities between [ca45 kDa] and [ca 23 kDa] by the MALDI-TOF MS method

noglobulin gammopathies was performed by MALDI-TOF MS analysis, immunoﬁxation (routine method),and SDS-polyacrylamide gel electrophoresis/WB/enhanced

coincided with those of FLCs in MM patients urine by gelelectrophoresis under non-reduction/WB/ECL detection.

chemiluminescence (ECL) detection and the methods wereIn Table 1a and 1b, the proﬁles of MALDI mass spectra

Fig. 1.

The data show FLC proﬁles in the urine of patientsʼ with monoclonal immunoglobulin gammopathy by MALDI-TOF
MS as well as immunoﬁxation and Western blotting. a) and b) indicate the BJP κ -types, and c) and d) are BJP
λ -types. The inset shows data on immunoﬁxation.
49June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Table 1a.

The proﬁles of MALDI mass spectra of commercially puriﬁed BJP κ - and λ -types
κ -typeMean

λ -type[ca 23 kDa]

[ca 45 kDa]

[ca 72 kDa]

[ca 95 kDa]

[ca 23 kDa]

[ca 45 kDa]

[ca 72 kDa]

[ca 95 kDa]

23621.347165.971272.2

94520.3

Mean

22515.4

45134.2

67696.090381.5

SD

47.7

71.3

684.9

252.0

SD

29.7

64.0

52.3

113.3

CV (%)

0.2

0.15

0.96

0.03

CV (%)

0.13

0.14

0.08

0.12

Table 1b.

The proﬁles of MALDI mass spectra of BJP κ - and λ -types derived from patients with deﬁnite MMs
κ -type

λ -type[ca 23 kDa]

[ca 45 kDa]

[ca 72 kDa]

[ca 95 kDa]

Mean

24151.947698.072480.1

95271.2

SD

803.9

1491.72271.31147.9

3.33

3.13

3.13

1.20

CV (%)[ca 23 kDa]

[ca 45 kDa]

[ca 72 kDa]

[ca 95 kDa]

Mean

22803.045667.967620.691643.0

SD

118.7

582.2

1243.51640.5

0.52

1.27

1.84

1.79

CV (%)of the commercially puriﬁed κ - and λ -types (1a), andthose of the corresponding FLCs of MM patients deﬁnitely
diagnosed with immunoﬁxation (1b) are listed. The
MALDI mass proﬁles (n＝10) of the commercially puriﬁedκ -type were m/z 23621.3＋/−47.7 Da, 47165.9＋/−71.3 Da,
71272.2＋/−684.9 Da and 94520.3＋/−252.0 Da, respectively. Those of the commercially puriﬁed λ -type were at m/z
22515.4＋/−29.7 Da, 45134.2＋/−64.0 Da, 67696.0＋/−
52.3 Da, and 90381.5＋/−113.3 Da. On the other hand,
Table Ib shows that the MALDI mass spectra of 18 cases ofthe κ -type in MM patients urine were m/z 24151.9＋/−
803.9 Da, 47698.0＋/−1491.7 Da, 72480.1＋/−2271.3 Da,
and 95271.2＋/−1147.9 Da, and those of 12 cases of the
λ -type were m/z 22803.0＋/−118.7 Da, m/z 45667.9＋/−
582.2 Da, m/z 67620.6＋/−1243.5 Da, and 91643.0＋/−
1640.5 Da. All of the series of the m/z of κ -types wereclearly separated and greater than those of the λ -types, theFig. 2.

same as clinical samples as well as commercially puriﬁed
standards. However, there will be a possibility of erroneous

The ratios of [ca 45 kDa] / [ca 23 kDa] in MM
patientsʼ urine were calculated from ion intensitiesof [ca 45 kDa] and [ca 23 kDa].
The data of [ca 45 kDa] / [ca 23 kDa] were 0.054＋/−0.06
at the κ -type (n＝26) and 0.628＋/−0.43 at the λ type (n＝12), and also examined the puriﬁed BJP λ -typewere and 1.160＋/−0.62 at l type (n＝10). The p-values
less than 0.001 were considered signiﬁcant between the
BJP κ -type versus BJP λ -type, and the κ -type versus
puriﬁed λ -type, but the p-value was 0.06, not signiﬁcat,
between the BJP λ -type versus puriﬁed λ -type.

identiﬁcation between the two types using only monomer
or divalent ions of FLCs due to their heterogeneities and/or
coexistence of low molecular proteins and so we applied todiscriminate the two isotypes of BJP with the ratios
between dimer and monomer ions.
As shown in Fig. 2, the ratios of ion intensities between [ca45 kDa] and [ca 23 kDa] in 38 cases of deﬁnite MM patientsurine were calculated from the relative ion intensities at m/z[ca 45 kDa] and [ca 23 kDa]. The mean ＋/−standard devia-

patients were 1.112＋/−0.616 at the λ -type (n＝10). The
p-values less than 0.001 were considered signiﬁcant between

tions of the BJP κ -type (n＝26) were 0.054＋/−0.053 andthose of the λ -type (n＝12) were 0.572＋/−0.448. We also

the BJP κ -type versus BJP λ -type and those of the κ -type versus puriﬁed λ -type was less than 0.0001, but the p-value wasrevealed that those of the puriﬁed λ -type of BJP from MM

0.06 between the BJP λ -type versus puriﬁed λ -type. As
50

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Table 2.

The frequency distributions of the ratios between [ca 45 kDa] and [ca 23 kDa]

The 38 cases with deﬁnitive MMG[ca 45 kDa]/[ca 23 kDa]
BJP-kappa type
BJP-Lambda type＜0.02 0.02‒0.04 0.04‒0.06 0.06‒0.08 0.08‒0.1 0.1‒0.2 0.2‒0.4 0.4‒0.6 0.6‒1.0 1.0‒2.0 ＞2.0
*13

2

1

1

6

3

0

0

0

0

0

0

0

0

0

0

2

2

3

2

3

0

*The numbers included the 7 cases which [ca 45 kDa] did not detect at MALDI mass spectra.
The 23 cases with suspicious MMG[ca 45 kDa]/[ca 23 kDa]

＜0.02 0.02‒0.04 0.04‒0.06 0.06‒0.08 0.08‒0.1 0.1‒0.2 0.2‒0.4 0.4‒0.6 0.6‒1.0 1.0‒2.0 ＞2.0
**12

1

2

0

2

3

2

0

1

0

0

**The numbers included the 8 cases which [ca 45 kDa] did not detect at MALDI mass spectra.

Fig. 3.

The MALDI mass patterns of BJP κ - and λ -types coexisting light chains derived from intact immunoglobulin in
urine of deﬁnitely diagnosed MM patients often detected doublet peaks at m/z [ca 23 kDa] as shown in Fig. 3a, 3c,and 3e. On the other hand, the patterns of only BJP κ - or λ -types always showed single and sharp ion peaks, as presented in Fig. 3b, 3d, and 3f.

shown in Table 2, the cut-oﬀ values of the ratios of [ca45 kDa] / [ca 23 kDa] for the discrimination between BJP κ -cut-oﬀs, 0.1, could be clearly discriminated between BJP κ and λ -types. Using the ratios of [ca 45 kDa] / [ca 23 kDa],and λ -types would be estimated as from 0.1 to 0.2. Addi-

rapid and clear discrimination between the BJP κ - and λ-types of MM patients with only 1μL of spot urine was pos-tionally, the ratios of [ca 45 kDa] / [ca 23 kDa] in 23 cases of
suspicious MMG patients urine were also calculated. The
cut-oﬀs in the suspicious MM would be also as from 0.08 tosible.
6)

7)

and B.M. Bailey et al. reported thatthe ratio of BJP κ - and λ -types in urines derived fromT. Nakano et al.

0.1. From these frequency distributions, we deﬁned that the51

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Medical Mass Spectrometry Vol. 5 No. 1

Fig. 4. The typical MALDI-TOF mass spectra of two severe proteinuria after treatment with only SpinFilter3K (Fig. 4a and 4b)TM
and additional treatment with Aﬃ-Gel (Fig. 4c and 4d).
Insets show the data on immunoﬁxation.

MMG patients by immunoelectrophoresis and/or immunoﬁxation for the diagnosis of Bence-Jones proteinuria. Theirelectrophoretic technique. On the other hand, the patternsof only BJP κ - or λ -types showed single and sharp ion

protocols essentially need two devices, one is an electro-

peaks, as presented in Fig. 3b, 3d, and 3f. The half width ofphoresis to separate the target proteins, and the other is an
immunodetection of the target protein with speciﬁc anti-

the [ca 23 kDa] mass spectra would be a quite diﬀerent
between the cases of BJP alone and those of the coexis-

body. Our protocol using MALDI-TOF MS technique don tneed any antibodies to target proteins in the clinical sam-tence of intact immunoglobulin, as shown in Fig. 3. DR.

ples.

also used immunoaﬃnity puriﬁcation targeting the
FLCs prior to the MALDI-TOF MS analysis. Indeed, the

21)

22)

Barnidge et al. , MC. Kohlhagen et al. , and P. Milani et
23)

al.

Fig. 3a, 3c and 3e show typical MALDI mass patterns of
BJP κ - and λ -types with the same coexistence of light

FLCs in patient s urine could be suﬃciently concentrated

chains derived from intact immunoglobulin G and A in

but the MALDI mass spectra were still interfered by theurine of deﬁnitely diagnosed MM patients. We often could

excessive of albumin. Our fundamental preparations areobserve doublet peaks at m/z ca 23 kDa, which overlappedwith the ion for the κ - or λ -types. The origin of the doublet

only desalting and ﬁltration using SpinFilter 3K

TM

without

any puriﬁcation with speciﬁc antibody.
Fig. 4. shows the MLDI-mass spectra of urinary protein

ion peaks indicated that one would be BJP itself and theother would be the light chain derived from an intact immu-

proﬁles with severe proteinuria with and without Aﬃ-GelTMnoglobulin. EM. Bailey et al. presented that the light chain

7)

Blue resin. Before preparation with the resin, multivalentladder pattern reﬂected the heterogeneity of normal lightchains. The authors indicated that the ladder pattern proba-ions of albumin, especially a trivalent ion at m/z ca
22.5 kDa, overlapped the ions of the κ - and λ -types. Afterbly reﬂects the limited heterogeneity of normal light chains

the preparation, the MALDI mass spectra of albumin completely disappeared and proﬁles of BJP- λ could be clariﬁed,

and so the presence of the ladder pattern in urine mightinterfere the detection of low levels of BJP sharing the electrophoretic mobility of one of the normal light chain bands.

as shown in Fig. 4c and 4d. In the present study, if the samTM
Blue resin
ple revealed severe proteinuria, Aﬃ-Gel

For this reason, the MS analysis has an advantage than thewould be used to remove excessive albumin in the sample,
52

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Medical Mass Spectrometry Vol. 5 No. 1

TM

21)

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