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INTRODUCTION

Inherited hemoglobin disorders are common worldwide

due to population migration. These disorders can be

characterised by mutations in the globin gene which may

result in a structural abnormality or a reduction in the

rate of synthesis of one of the globin chains. A structural

change may be harmless, however, some may impair

function of hemoglobin and oxygen transport. Here, we

will describe a SONAR quadrupole scanning data

independent acquisition (DIA) method for the precise

identification of several variants that may be encountered

in either the α- or β-hemoglobin chains.

Hemoglobin (Hb) variants are currently detected using cation-

exchange high performance liquid chromatography (HPLC) or

isoelectric focussing (IEF) separation techniques. These

methods are able to flag the presence of Hb variants but are

unable to provide conclusive identification of any variant.

Definitive characterisation requires protein sequence

elucidation using mass spectrometry or DNA analysis. The

identification of Hb variants using mass spectrometry is

generally performed in three steps: (1) detection of the type of

globin chain variant at the intact level, (2) identification of the

variant peptide following tryptic digestion and (3) sequence

analysis of the variant tryptic peptide by tandem mass

spectrometry [1]. In order to minimise sample preparation and

analysis times, all the steps in this procedure involve no

chromatographic separation of components prior to analysis.

A Novel Data Independent Acquisition Method for Hemoglobin Variant Identification in Clinical Research

Jonathan P. Williams1; Christopher J. Hughes1; Heather A. Brown1; Keith Richardson1; Johannes P.C. Vissers1 and LeRoy B. Martin2

1Waters Corporation, Wilmslow, United Kingdom; 2Waters Corporation, Beverly, MA, USA

Figure 1. Structure of hemoglobin: α2β2 (+ 4 heme groups).

METHODS

Sample preparation and SONAR MS

A 50-fold diluted blood sample was digested with trypsin for 30 minutes.

The mixture of tryptic peptides was further diluted 10-fold for direct

infusion SONAR-MS analysis. The samples were infused using a

Harvard Apparatus (South Natick, MA, USA) Model 22 Syringe Pump at

a flow rate of 10 µL/min as shown In Figure 2.

Mass spectrometric analysis was performed using a Xevo G2-XS QTOF

mass spectrometer. Data acquisition and processing was carried out

using MassLynx v4.1. Instrumental parameters used during the study

were:

SONAR-MS: Xevo G2-XS

Ion mode: ESI positive

Capillary: 2kV

Source Temp: 110oC

Desolvation Temp: 250oC

In SONAR mode the quadrupole was rapidly and repetitively

scanned over an m/z range that included both the normal and variant

tryptic peptides. The quadrupole transmission window was optimised

between 0.5-5Da depending on the variant. The oa-TOF records mass

spectra as the quadrupole scans and stores the MS data in 200 discrete

bins. Two data functions were acquired in an alternating fashion,

differing only in collision energy. In the low energy MS1 mode, data

were collected at a collision energy of 6 V and at an optimised value

between 18 V and 35 V in the elevated energy MS2. The resulting data

set contains both precursor ions and all associated product ions. The

spectral acquisition (and quadrupole scan) time in each function was 0.5

s.

Data processing and database searching

The resulting 2D SONAR data were processed within Driftscope.

Informatics typically employed for discovery proteomics experiments

were further refined and used for automated processing for precise

variant identification. SONAR Hb variant data were peak detected and

processed using Skyline open source informatics components that were

automated using Symphony, a client/server application that was

triggered by the data acquisition system. An example of the Symphony

interface is shown in Figure 3. Skyline was further used to screen for a

number of unique Hb variant peptides and demonstrate the feasibility of

Hb variant identification using discovery bio-informatics tools in

combination with SONAR acquisition.

RESULTS AND DISCUSSION

Adult Hb variants, as investigated here are often detected as part of a

routine glycohemoglobin or antenatal screening healthcare programme.

The three step procedure involves sequencing peptides via low-energy

collision-induced dissociation (CID) using a tandem quadrupole

instrument and >95% of the variants encountered in practice can be

identified using this procedure. There are a number of variants which

involve amino acid exchanges governed by single mutations in the

nucleotide codon to either leucine or iso-leucine that cannot be

differentiated using this procedure [2]. Here ,we demonstrate a SONAR

data independent acquisition (DIA) approach for precise

hemoglobinopathy characterisation. A number of variants that were

encountered in either the α- or β-hemoglobin chains were investigated

as shown in Table 1.

Table 1. Human Hemoglobin heterozygous variants investigated during

this study.

(i) Study of Hb Sickle (β6 Glu→Val), -30 Da

Figure 4 shows a sample SCX chromatogram and a peak eluting after

the wild-type adult Hb (A0, 2.49 minutes). The elution time of this peak

(4.37 minutes) falls within the Sickle window and usually corresponds to

the Sickle variant. A Sickle solubility test can confirm this and precise

identification can be achieved easily using MS.

Figure 4. Cation-exchange HPLC trace of an abnormal blood sample.

Sample Variant Mutation Mass change (Da) MS/MS

1 Old Dominion β143 His→Tyr +26 Yes

2 J-Norfolk α57 Gly→Asp +58 Yes

3 Sickle + G-Philadelphia α68 Asn→Lys +14 No

4 G-San Jose β7 Glu→Gly -72 Yes

6 Osu-Christiansbourg β52 Asp→Asn -1 Yes

7 Q-India α64 Asp→His +22 Yes

8 Winnipeg α75 Asp→Tyr +48 Yes

9 J-Meerut α120 Ala→Glu +58 Yes

10 O-Arab β121 Glu→Lys -1 No

11 Bethesda β145 Tyr→His -26 No

12 Korle-Bu β73 Asp→Asn -1 Yes

13 Sickle β6 Glu→Val -30 Yes

14 D-Punjab β121 Glu→Gln -1 No

15 D-Iran β22 Glu→Gln -1 Yes

16 C β6 Glu→Lys -1 No

17 E β26 Glu→Lys -1 No

18 South Yorkshire α50 His→Tyr +26 Yes

Driftscope visualisation quadrupole m/z vs. TOF m/z

Fig. 5(C)- 2D data file of quadrupole m/z vs fragment mass

showing intensity, generating a tandem MS spectra for both

normal and variant ions for the heterozygote samples. Tan-

dem MS is usually required due to the possibility that more

than one amino acid could give rise to mutation. For exam-

ple, 3 amino acids could mutate to give a –30 Da mass de-

crease from normal within the βT1 peptide (Fig. 4 inset). *NL

-not listed.

Figure 5. Part-digest MS spectrum of a Sickle heterozygote

showing normal (A) and variant ions βT1+ ions (B).

Driftscope 2D plot of quadrupole m/z vs. TOF m/z (C) where

the quadrupole was scanned linearly from m/z 400-500 with

an isolation width of 2 Da. Digest SONAR tandem MS spec-

trum of a normal Hb (D) and Sickle heterozygote (E). Note:

yʺ2 is detected at m/z 246 in both spectra which shows that

position 7 has not mutated. b4 detected at m/z 451 in both

spectra which shows that position 4 has not mutated. b6 in

normal detected at m/z 677 and detected at m/z 647 in vari-

ant and thus confirms sickle heterozygote.

(iii) Study of Hb D-Iran (β22 Glu→Gln), -1 Da

(ii) Study of Hb South Yorkshire (α50 His→Tyr), +26 Da

(iv) Bioinformatics

Infusion SONAR-Hb variant data were peak detected and processed

using Skyline Runner components that were automated using Sym-

phony. In brief, Symphony automation pipelines combine a list of tasks

that are executed upon input data. Symphony provides means to cor-

rectly configure and combine tasks and set up so called pipeline defini-

tion files. The pipeline example shown in Figure 2 combines a data

transfer step with remote Skyline processing on a network connected

PC and optional Panorama upload.

Skyline was used to screen for the variants listed in Table 1 against their

unique Hb variant peptides to demonstrate the feasibility of Hb variant

identification using discovery bio-informatics tools in combination with

SONAR acquisition. The results panels in Figure 8 show the possible

detection of the ‘D-Iran’ Hb variant in one of the investigated samples as

indicated by the average reduced mass errors compared to the other

samples and the number of detected product ions and the positional

identification of the position of the βeta chain mutation.

(i) Study of Hb Sickle (β6 Glu→Val), -30 Da (cont.)

SONAR was employed since more than one amino acid within the αT6 peptide could mutate to give a 26Da mass in-crease from normal. The mutation Ser→Leu/Ile is highly unlikely even before tandem MS is performed based on the number of base changes required for this mutation to occur. The digest accurate mass MS spectrum can also rule out Ala→Pro. Thus, 2 possibilities exist namely His→Tyr at posi-tions 45 and 50 in the α-chain. Figure 6. Part-digest mass spectrum of a Hb South Yorkshire

showing normal (A) and variant ions αT62+

ions (B).

Driftscope 2D plot of Quadrupole m/z vs TOF m/z (C) where

the Quadrupole was scanned linearly from m/z 900-1000

with an isolation width of 2Da. Digest SONAR tandem mass

spectrum of a normal Hb control (D) and the variant Hb (E).

Note: yʺ6 is detected at m/z 589 in both spectra. A 26Da

mass increase observed at yʺ7 confirms Hb South Yorkshire.

SONAR was employed to distinguish βT3 peptide mutations that could indicate a 1 Da mass decrease from normal. Some examples included in the Hb Var Database include β21 Asp→Asn (Hb Cocody), β22 Glu→Gln (D-Iran), β22 Glu→Lys (E-Saskatoon), β26 Glu→Gln (King’s Mill), β26 Glu→Lys (Hb E). The Glu→Lys mutations can be discounted since these will produce a new cleavage point, hence the for-mation of 2 new peptides. Figure 7. Part-digest mass spectrum of a Hb D-Iran showing

normal (A) and variant ions βT3+ ions (B). Driftscope 2D plot

of Quadrupole m/z vs TOF m/z (C) where the Quadrupole

was scanned linearly from m/z 600-700 with an isolation

width of 0.5Da. Digest SONAR tandem mass spectrum of a

normal Hb control (D) and Hb D-Iran (E). Note: yʺ7 is de-

tected at m/z 659 in both spectra. A 1 Da mass decrease ob-

served at yʺ9 confirms Hb D-Iran.

Figure 7. Skyline detection of SONAR data of Hb variants and the

identification of Hb D-Iran (y9 886.4741).

CONCLUSIONS

SONAR technology precisely identified a number of Hb variants

The results show that a rapidly scanning quadrupole analysis method can quickly process peptide digests from whole blood without chromatography

Further informatics tools would need to be developed to fully automate the procedure for large scale high throughput unknown variant identification

References

1. B. Wild et al. Rapid identification of hemoglobin variants by

electrospray ionization mass spectrometry. Blood Cells Mol. Dis. 27

(2001) 691-704

2. J. P. Williams et al. Hot electron capture dissociation distinguishes

leucine from isoleucine in a novel hemoglobin variant, Hb Askew,

βeta 54(D5)Val -> Ile. JASMS. 20 (2009) 1707-1713

Acknowledgements

We would like to thank Deborah Mantio, Central Middlesex Hospital, London for the blood samples.

For Research Use Only.

Not for use in diagnostic procedures.

Figure 3. The pipeline example shown combines a data transfer step

with remote Skyline processing on a network connected PC and

optional Panorama upload.

A0, WT A0,

Sickle

Figure 2. Experimental workflow infusion SONAR for Hb variant

analysis.