utilizing proteomics to characterize hair and hair damage

UTILIZING PROTEOMICS TO
CHARACTERIZE HAIR AND HAIR
DAMAGE AT THE MOLECULAR LEVEL
P1316
Michael G. Davis 1 , Michael J. Flagler 1 , Stephen W. Hendrix 1 , Yiping Sun 2 ,
Tanuja Chaudhary 2 and Kenneth D. Greis 3
1
P&G Beauty, Cincinnati, OH, USA; 2 P&G Global Analytical, Cincinnati,
OH, USA; 3 University of Cincinnati, Cincinnati, OH, USA
This work was funded by P&G Beauty & Grooming

INTRODUCTION
• Proteins are the major
structural component
of hair (~80-90%)
Component
Protein
Lipid
Protein bound sulfur
Sugars
Ash
Zinc
Melanin (black hair)
Approximate content
by dry weight of hair
91%
4%
4.7%
1.0%
0.5%
200 ppm
4%
Feughelman et al. J. Appl. Polm. Sci., 2002, 83,489.
• Hair is not regenerative after it exits the scalp; therefore, maintaining
proper hair architecture at the molecular level is an important hallmark for
a healthy hair structure
• Ultimately, proper hair protein structure has an important impact on the
cosmetic value of the hair shaft

PURPOSE/OBJECTIVES
• Develop new methods and models for hair shaft
damage and abnormalities to drive new
understanding at protein level and achieve more
sensitive diagnostics for early detection of damage
• Apply these methods to provide insights into how the
molecular structure of the hair shaft changes in
response to different insults

METHODS
• Traditional Protein loss: 0.2-0.3g hair samples (2in. length) were added to glass
scintillation vials. Distilled water was added at a ratio of 10:1 (water to hair). Samples were
shaken for timepoints up to 1h at 2,500 rpm on a DVX-2500 Multi-2 Vortexer platform
(VWR). Water extracts were removed from vials and protein concentration determined using
the Modified Lowry assay (Pierce).
• Soluble/Insoluble Protein Loss: Samples following traditional protein loss method (above)
were subjected to centrifugation at 14,000 rpm to separate into soluble/insoluble fractions.
Pelleted material (insoluble) was solubilized in 3M urea, 1M NaOH, 0.06% CHAPS followed
by sonication for 30 min in a Branson B300 sonicating water bath (34KHz). Protein
concentration in soluble/insoluble fractions was determined using the Modified Lowry assay.
The insoluble protein fraction was shown by SEM to consist of cuticle, whereas the soluble
fraction was shown by mass spec to consist primarily of cortex proteins.
• 2D Gel Electrophoresis: Hair samples pre- and post-bleaching (approx. 250mg) were
clipped into 1mm pieces and ground in a mortar and pestle pre-cooled with liquid nitrogen.
Samples were then extracted in 8M urea, 0.05M Tris, 0.05M DTT, pH 9.3 with end-over-end
mixing for 24h at RT. Following extraction, samples were centrifuged to pellet remaining hair
and the soluble fraction was subjected to reduction/alkylation and dialyzed twice against
distilled water. Following dialysis, samples were mixed 1:1 with 9M Urea, 4% CHAPS,
0.19% SDS, 2% DTT) and subjected to 15 min of ultrasonic treatment in sonicating water
bath (Branson 2510) to resuspend insoluble material. 25µg of protein was loaded into
immobilized pH gradient strips (pH 4-7) and separated using standard gel electrophoresis
techniques.
• MALDI-TOF: Water extracts from protein loss method were analyzed by MALDI-TOF/TOF
using standard protocols.

METHODS: AN OVERVIEW
MW
pI
2D gels
(of hair fiber
sample)
Remove Hair
Soluble Protein Loss
(mainly cortex)
Centrifuge
Pellet
Insoluble Protein Loss
(cuticle)
Spot directly in matrix
Hair Sample
Proteins Eluted
in water
Protein concentration
analyzed
Traditional
Protein Loss
Laser
+
+
+
+
+
MS
MALDI-TOF Mass Spec

RESULTS: ALL HAIR LOSES PROTEIN
• Protein loss can be detected
after just 5 minutes of exposure
to water
• Protein loss increases with time
• Chemically damaged hair loses
more protein than virgin hair
• Measuring protein loss provides
early detection of damage,
before it’s too late
Protein Loss (ug/ml)
350
300
250
200
150
100
50
0
5
10
15
Virgin
Bleached
20
30
40
50
60
5
10
15
20
30
40
50
60
Extraction Time (min)
• Protein loss at 60 minutes is equivalent to
approx. 0.1% of total protein from virgin
hair and 0.4% from bleached hair
(measured by Modified Lowry assay)

PROTEIN LOSS CORRELATES WITH A MARKER
OF OXIDATIVE DAMAGE
Protein Loss (µg/ml)
1500
1000
500
0
Virgin
MIld Bleach
Heavy Bleach
Hair Type
Description
Cysteic Acid
(units)*
Virgin 26.8
Mild Bleach
(Low Lift)
Heavy Bleach
(High Lift)
53.4
155.3
* A measure of oxidation of surface cystine; measured
by FTIR-ATR.
• Protein Loss, as measured by the Lowry assay, increases with the level of
chemical damage and correlates with cysteic acid formation, a well known marker
for disulfide breakdown during the bleaching process

NOT ALL HAIR DAMAGE IS THE SAME: DIFFERENTIAL
PROTEINS ARE TARGETED BY DIFFERENT INSULTS
• Separation of soluble and insoluble protein loss indicates that bleaching targets predominantly
insoluble proteins (cuticle)
Bleached SOLUBLE
Bleached INSOLUBLE
400
400
Protein Loss (µg/ml)
300
200
100
Protein Loss (µg/ml)
300
200
100
0
0
100
Untreated
Bleached
• Contrarily, exposure of hair to UV results in loss of almost exclusively soluble proteins (mainly
cortex)
UV SOLUBLE
UV INSOLUBLE
100
Untreated
Bleached
Protein Loss (µg/ml)
50
Protein Loss (µg/ml)
50
0
Untreated
40 hours
0
Untreated
40 hours
Data expressed as Mean + SEM

FRAGMENTATION OF KERATIN 31 IS A BIOMARKER
OF CHEMICAL OXIDATIVE DAMAGE
• MS analysis has shown that keratin 31 is cleaved at the same sites to generate the
same peptide fragments every time hair is bleached
4700 Reflector Spec #1 MC[BP = 834.9, 285]
% % Intensity
100
90
80
70
60
50
40
30
20
10
800.2531
824.8885
834.8649
844.3223
839.2581
881.0795
886.2781
916.3190
956.3007
993.8842
987.2777
1036.2694
1177.2810
1278.4763
1405.4078
Virgin Hair
284.6
% %
Intensity
0
799.0 1040.8 1282.6 1524.4 1766.2 2008.0
100
90
80
70
60
50
40
30
20
10
807.1583
850.1978
879.1765
916.1785
969.1910
994.2286
998.1843
1038.1949
1042.2292
4700 Reflector Spec #1 MC[BP = 994.2, 1100]
1061.1953
1085.2305
1119.2626
1148.1992
1188.2072
1205.1962
1229.2646
1278.4066
1308.2894
Mass (m/z)
Mass (m/z)
Keratin 31
1342.2939
0
799.0 1040.8 1282.6 1524.4 1766.2 2008.0
Mass (m/z)
(m/z)
1387.3157
1430.3033
1470.3545
1499.3151
Bleached Hair
1529.3547
1581.3235
1596.3389
1639.3330
1642.3396
1684.3221
1715.3872
1760.4307
1794.3939
1830.4374
1873.3491
1908.4258
1941.4189
1979.3434
1100.4

FURTHER MS FOLLOWING TRYPTIC
DIGESTION
• Full view of protein loss with tryptic digest for bleached hair reveals
more than just keratin 31 but also other structural keratins within the
cortex
Keratin
Fragment
+/- Tryptic
Digest
Acidic/Basic
Cortex/Cuticular Origin
Keratin 31 - Acidic Cortex
Keratin 81 + Basic Cortex
Keratin 33a + Acidic Cortex
Keratin 33b + Acidic Cortex
Keratin 83 + Basic Cortex
Keratin 85 + Basic Both
Keratin 86 + Basic Cortex

2D GELS CONFIRM THE LOSS OF SPECIFIC
PROTEINS WITH BLEACHING
• 2D gels of hair samples pre- and post-bleaching confirms the loss of
keratins detected in water extracts, as well as keratin-associated proteins
kDa
188
98
4
Keratin 86
Virgin
pH
Keratin 85
Bleached
7 4 pH
7
Keratin 86 (missing)
Keratin 85 (less abundant)
62
49
Keratins
38
28
17
14
6
3
KAP 3.2
KAP 3.3
KAP
3.1
KAP
3.1
Keratin 31
KAP
3.1
KAP 3.2
KAP 3.3
Keratinassociated
proteins (KAPs)
KAP 3.2
KAP 3.3
KAP
3.1
KAP
3.1
Keratin 31
KAP
3.1
KAP 3.2
KAP 3.3
Keratin-associated
proteins (KAPs)
(missing)

SUMMARY
• Protein loss from hair increases as a result of damage and is
consistent with the level of damage to the hair shaft
• Protein loss correlates with other measures of hair damage
• Protein loss can be separated into soluble and insoluble
fractions, which differentially segment with particular damage
insults (UV vs. bleach)
• Specific sites in the protein structure of the hair are targeted by
oxidative chemistry resulting in peptide biomarkers of damage

CONCLUSIONS
• We have developed robust methods to examine hair proteins by
using state of the art proteomic techniques
– These techniques have started to give us a glimpse into hair
damage at the molecular level by examining the specific
proteins
• These methods provide a better understanding of structural
changes to the hair at the molecular level which could lead to
earlier detection of damage and new treatment opportunities
• Early intervention could help to prevent the negative tactile and
behavioral changes commonly associated with hair coloring