Human Hair Keratin Extraction and 

Genetically Variant Peptide Detection

Meghan C. Burke, Zheng Zhang, William E. Wallace, 

Yuxue Liang, Sergey Sheetlin, Yuri A. Mirokhin, 

Dmitrii V. Tchekovskoi & Stephen E. Stein
Human Hair-derived Genetically 

Variant Peptides (GVPs)

• Why hair?

• Why protein?

• Why peptides?

• Why GVPs?

 Chemically stable, easily obtained

 Chemically more stable than DNA

 Can provide inheritance information in the absence of high 

quality DNA

 Advantageous for complex mixtures and limited 

sample

 Ionize and fragment well in a mass spectrometer

Sequence Database 

Search Space

Spectral Library 

Search Space
Human Hair Structure

Bengtsson, C. F.; Olsen, M. E.; Brandt, L. Ø.; Bertelsen, M. F.; Willerslev, E.; Tobin, D. J.; Wilson, A. S.; Gilbert, M. T. P., DNA from 

keratinous tissue. Annals of Anatomy - Anatomischer Anzeiger 2012, 194 (1), 17-25.

Hair Root Hair Shaft
Protein Extraction From 

Human Hair

[1] Urea & Protease Max1

[2] NaOH & Sodium 

Dodecyl Sulfate (SDS)2

– Repeat extraction 3x

[3] Single Step Method 

(SDS Alone)

https://kimberlybiochemist.wordpress.com

1Parker GJ, Leppert T, Anex DS, Hilmer JK, Matsunami N, et al. 

(2016) PLOS ONE 11(9): e0160653.

2Wong, S. Y., Lee, C. C., Ashrafzadeh, A., Junit, S. M., Abrahim, N., & 

Hashim, O. H. (2016). PLOS ONE , 11(10), e0164993. 

Protease Max
Optimization of Single Step Method 

Protein Extraction

MW

Std (kDa)

Keratins

type II

type I

5 10 15 30 60 90

Time (min)

Time course study to optimize the heating time

Scanned Intensity

Time(min) Type I Type II

5 1582 3511

10 31593 34084

15 33281 36510

30 45417 53283

60 44089 51787

90 39515 45820

260

160

110

80

60

50

40

30

20
Reproducibility of Extraction Methods

NaOH & SDS Extraction Method Single Step Extraction Method

Summary: 

Single step extraction method is simpler and more reliable regarding 

the sample preparation

1A

MW

Std (kDa) 1B 1C 1D 1E 1F 1G 1H

type II

type I

1 hair (5 cm)

10 hairs (5 cm)

1 hair (5 cm)

MW

Std (kDa) A B C D E F G H

Keratins

type II

type I

Keratins

260

160

80

60

50

40

30

20

110

260

160

80

60

50

40

30

20

110
Sensitivity Across Three Extraction 

Methods
Sensitivity Across Three Extraction 

Methods

Hair Length (cm) Dilution Fold

Convert Serial Dilution to Hair Length Equivalent

Protease Max

NaOH & SDS

Single Step
Comparison of Experimentally 

Introduced Modifications
Single Step Extraction

Reduction of Length of Human Hair

SDS-PAGE

In-gel 

digestion Keratins

5 2.5 1cm

MW

Std (kDa)

Hair 

Length

Proteome Hair Keratins

Proteins Peptides Proteins Peptides

5 cm 194 2224 15 740

2.5 cm 119 1578 14 663

1 cm 113 1546 14 561

Summary: 

• Similar results were obtained from 5-cm and shorter hair samples

• Starting material can be as little as 1-cm hair shaft

type II

type I

160

80

60

50

40

30

20

110
Mass Spectral Library Identification of 

Keratin-derived GVP

 

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140

0

50

100

50

100

175.1195

y1 175.1192

230.0779

b2 230.0775

284.1250

284.1245

413.1682

413.167

515.3317

y4 515.3307

644.3747

y5 644.3737

757.4590

y6 757.4579

886.5021

y7 886.5007

985.5709

y8 985.5695

y9+i 1100.6016

1110.5826

DNAELENLIR, Ala82→Val, Gene=KRT31

m/z
Summary

• The NIST single step protein extraction 

method provides best depth of coverage of 

hair proteome

• Identification of artifactual modifications 

reduces false positive GVP identifications

• Previously published GVP-containing peptides 

have been curated in a spectral library that 

will be publicly available

Contact: 

meghan.burke@nist.gov