US20110281256A1 - Systems and Methods of Detecting and Demonstrating Hair Damage Via Evaluation of Protein Fragments - Google Patents
Systems and Methods of Detecting and Demonstrating Hair Damage Via Evaluation of Protein Fragments Download PDFInfo
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- US20110281256A1 US20110281256A1 US13/109,546 US201113109546A US2011281256A1 US 20110281256 A1 US20110281256 A1 US 20110281256A1 US 201113109546 A US201113109546 A US 201113109546A US 2011281256 A1 US2011281256 A1 US 2011281256A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
- H01J49/0036—Step by step routines describing the handling of the data generated during a measurement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4742—Keratin; Cytokeratin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
- G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
Definitions
- Embodiments of the present disclosure are directed to a process for measuring the damage to the hair by the evaluation and identification of extracted protein fragments.
- Protein loss may be caused by everyday occurrences and environmental factors such as UV ray exposure, bleaching, coloring, perming, straightening, mechanical manipulation, and salt water contact.
- Proper hair architecture at the molecular level is an important characteristic of hair that has a healthy look, shine and feel.
- the hair comprises mostly protein and is not regenerative after it exits the scalp. Therefore, it is valuable to have products which protect the overall protein integrity of the hair. Thus, protection of the hair shaft on the protein and fiber level is important to ensure hair has a healthy look.
- Identifying the protein fragments extracted from the hair and correlating the type of protein fragment with a type of hair damage 1) enables a correct identification of the type of damage to the hair, and 2) may provide the information necessary to design products which either prevent the damage, or in the case of bleaches and/or other composition do not generate the damage. Additionally, it is also valuable to identify particular types of hair disease. Hair of individuals with hair diseases, do not react to damage and/or treatments in the same way as normal hair. Therefore, it may be possible to indicate what type of hair disease is present based upon the response of the hair at a protein level to a particular type of damage.
- the present disclosure relates generally to systems and methods for detecting types of hair damage by correlating protein fragments extracted from the hair to a type of hair damage.
- a method of correlating hair damage type or source to marker protein fragments comprising: generating two identical hair samples; sample A and sample B; applying a damaging composition or treatment to sample A; apply no damaging composition or treatment to sample B; extracting the labile proteins using a suitable solvent samples from each of sample A and sample B; analyzing the protein fragment samples from sample A and sample B with MALDI-MS; comparing the MALDI-MS results from sample A and sample B; identifying the marker protein fragments by identifying the unique modification patterns which exist in sample A that do not exist in sample B.
- a method for demonstrating hair damage type or source comprising: extracting the labile proteins using a suitable solvent from a hair sample; analyzing the protein fragments sample with MALDI-MS; resulting in protein fragment results; and identifying the hair damage by comparing the protein fragments results to a list of marker protein fragments for particular damage.
- FIG. 1 This figure is the MALDI-MS results of marker protein fragments for hair damaged by bleach in comparison to the MALDI-MS results of protein fragments for undamaged hair.
- hair means keratinous fibers of the human or animal origin, such as hairs on the head or eyelashes.
- keratinous protein is understood to mean those proteins present in hair.
- protein fragments means the amino acids and larger proteins that are damaged and broken off the keratinous protein structure and held within the hair structure by electrostatic interactions, weak hydrogen bonding matrix proteins and lipids, or any other force that does not include incorporation in the keratinous protein structure.
- marker protein fragment means the protein fragment which has been correlated to a particular type of hair damage and/or damaging treatment.
- elutes means removing proteins from hair via contacting hair with an aqueous solution without the addition of any reduction or extraction agents, thereby yielding no modification of the keratinous protein structure and no breaking or reduction of chemical bonds present in the hair sample other than electrostatic interactions, weak hydrogen bonding matrix proteins and lipids, or any other force that does not include incorporation in the keratinous protein structure.
- “elutable” means protein fragments present in the hair sample that may be removed from the hair structure in an aqueous solution without the addition of any reduction or extraction agents. Furthermore, “elutable” means proteins that may be carried out of the hair structure in an aqueous solution consisting essentially of water without the breaking or reduction of chemical bonds present in the keratinous protein structure other than electrostatic interactions, weak hydrogen bonding matrix proteins and lipids, or any other force that does not include incorporation in the keratinous protein structure.
- a method for detecting and demonstrating hair damage by utilizing an aqueous solution to extract protein fragments from the hair without modifying the keratinous protein structure is described in Patent Application No. 61/345,321 filed on May 17, 2010.
- the protein fragments are analyzed. From the analysis of the protein fragments it is possible to identify the type of damage that has been done to the hair, in particular it is possible to determine the source of the damage to the hair.
- One such specific marker protein fragment includes those marker protein fragments generated when the hair is bleached.
- a hair sample can be tested, the protein fragments extracted, and the resulting protein fragments tested using an antibody based detection, and/or a mass spectrometry technique.
- the protein fragments are evaluated using the Matrix Assisted Laser Desorption Ionization (“MALDI”), also known as the MALDI-TOF Mass Spectrometry “MALDI-MS”.
- MALDI-MS Matrix Assisted Laser Desorption Ionization
- MALDI-MS can be used for the analysis of biomolecules such as peptides and proteins and large organic molecules such as polymers.
- the analyte is first co-crystallized with a UV absorbing matrix such as alpha-cyano-4-hydroxycinnamic acid (CHCA), then subjected to pulse laser (YAG or nitrogen laser) radiation.
- CHCA alpha-cyano-4-hydroxycinnamic acid
- MALDI-TOF a time-of-flight mass analyzer is used.
- MALDI-TOF Data can be acquired in MS mode to generate molecular weight information (e.g., a peptide) and in MS/MS mode (e.g., a peptide sequence/structure information).
- Typical MALDI mass spectrum acquisition takes less than a minute so it can be used for fast screening of molecular species in samples of interest. Changes and molecular makers can be detected by comparing the mass spectra acquired in samples treated under different conditions such as virgin hair vs. bleached hair.
- MALDI-MS can be performed either with or without enzymatic digestion of proteins.
- the protein fragment test results are then compared to a library of known marker protein fragments to identify what type of hair damage, and in some situations, what is the original source of damage to the hair i.e. bleach. This enables a “fingerprinting” of damage; meaning that if a hair sample is tested and the results include certain marker protein fragments, then the hair sample has been damaged by a particular source.
- Additional methods for evaluating the protein fragments include, but are not limited to, liquid chromatography-electrospray mass spectrometry, antibodies against the protein fragments could be generated and an ELISA assay could be developed.
- reagents available through Applied Biosystems, Carlsbad Calif. can be used to establish covalent amine linkage of an isobaric tag to each lysine side chain and free N-terminal group of a protein fragment. This allows for multiple samples to be run simultaneously through the MALDI MS. Running multiple samples through the MALDI MS simultaneously minimizes variations in the test data due to test variability.
- a library of these marker protein fragments can be generated by damaging swatches of hair with a variety of different compositions or treatments and then analyzing the resulting protein fragments in comparison with a similar swatch of hair which has not been damaged.
- Marker protein fragments can be identified by the MALDI-MS, as it is believed the same marker protein results will be found based upon the type of damage that the hair has experienced. This means that the marker protein fragment is indicative of a type or source of hair damage. Hair damages by bleach results in particular marker protein fragments, hair damaged by UV results in particular marker protein fragments etc.
- the additional peaks result form chemical modification at the amine termini of the fragments.
- the protein fragments that correspond to the characteristic peaks are sequenced (see Table 1). Letters used to denote the protein sequences refer to the one-letter amino acid code (i.e.
- E glutamic acid
- I isoleucine etc.
- Additional modifications to these proteins include oxidation at Methionine, cysteine, and tryptophan residues, and deamindation at asparagine and glutamine residues can also be detected.
- the protein fragments mapped to Keratin 31, suggesting that this protein is degraded upon bleaching of the hair shaft.
- this method can also be used to indicate whether an individual's hair has a normal response to treatments. For example if an individual has a particular hair disease, a hair sample from this individual may not generate the same marker protein fragment as would a person who has a “normal” response. A person with a hair disease may generate additional, less and/or even different marker protein fragments than would be indicated by a normal response.
- a library of hair disease responses could also be created similar to that of the marker protein fragments for damage as described above. Therefore, a test for this hair disease could include exposing an individual's hair to a particular damaging treatment and then identifying the hair disease by the marker protein fragments that are generated from the damaging treatment.
- the soluble and insoluble protein fragments are analyzed separately. Analyzing the soluble and insoluble protein fragments separately can result in higher sensitivity of protein fragment detection. Additionally, analyzing these protein fragments separately may further refine the determination of the location of the damage to the hair. To measure the soluble and insoluble protein fragments separately, after removal of the hair fibers the sample the in water can be centrifuged or the insoluble portion can be left to settle out from the soluble portion.
- a bleaching solution consisting of 2% ammonium hydroxide, 0.2% tetrasodium EDTA (pH adjusted to 10.3 with acetic acid), and 6% hydrogen peroxide was prepared. Hair tresses (brown and natural white) were submerged in the bleaching solution and placed in a 40° C. oven. At timepoints of 30 to 90 minutes, hair tresses were removed from the bleaching solution, washed under DI tap water for two minutes, and dried.
- the amount of overall protein damage sustained by the hair tresses as a result of the bleaching treatments was assessed by measuring total protein loss from the hair. Briefly, 0.2-0.3 g of hair from each tress was clipped into 2 inch segments and added to a glass scintillation vial. DI water was added at a ratio of 1.0 ml DI water to 0.1 g hair, and samples were subjected to physical agitation for 60 minutes at 2,500 rpm on a vortex platform. Water extracts were analyzed for total protein concentration using the Lowry protein quantification assay. Results are summarized in Table 2.
- the specific protein damage sustained by the hair tresses as a result of the bleaching treatments was determined by analyzing the water extracts described above by MALDI-TOF analysis.
- Water extracts were mixed (1:1) directly with a MALDI matrix solution (5 mg of a-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in 1 ml of 80:20:0.1 acetonitrile: water: trifluoroacetic acid). About 1 ul each sample was spotted on the MALDI plate and analyzed by MALDI-TOF/TOF 4800 plus system (AB-Sciex).
- Protein markers for hair damage detected in the water extracts of bleached hair include peaks with m/z 994/1037, 1596/1639, 1204, 1278 etc. Note peak 1038 is the deamidated form of 1037 and delta mass is 43 Da among the peaks 994/1037 and 1596/1639, etc. Results are summarized in Table 1 for the markers 1037 and 1204.
- a bleached water extract were separated by a reversed phase HPLC (2 mm ⁇ 15 cm, Jupiter, C4, 300 A column, HP1100 system), fractions were collected manually and lyophilized. Each fraction was re-dissolved in 20 ul of 0.02% trifluoroacetic acid (TFA)/water solution, 1:1 mixed with the MALDI matrix, spotted on MALDI plate for MALDI-sequencing.
- TFA trifluoroacetic acid
- another water extract of bleach hair was dried under vacuum and re-dissolved in 50 mM NH4Ac buffer (pH 8).
- Hair tresses (General Population brown hair) were exposed to UV light for time points of up to 75 h in an Atlas Ci3000+Xenon Arc Fade-Ometer at an irradiance setting of 1.48 W/m 2 at 420 nm, a chamber temperature of 35° C., and 80% relative humidity.
- One hour of UV exposure under the these conditions is approximately equivalent to 7.5 hours of external sun exposure in Florida, according to calculations performed using an Outdoor to Xenon Radiant Energy Conversion program provided by the manufacturer (Atlas Material Testing Technology LLC).
- the amount of overall protein damage sustained by the hair tresses as a result of the UV exposure was assessed by measuring total protein loss from the hair. Briefly, 0.2-0.3 g of hair from each tress was clipped into 2 inch segments and added to a glass scintillation vial. DI water was added at a ratio of 1.0 ml DI water to 0.1 g hair, and samples were subjected to physical agitation for 60 minutes at 2,500 rpm on a vortex platform. Water extracts were analyzed for total protein concentration using the Lowry protein quantification assay. Results are summarized in Table 3.
- the specific protein damage sustained by the hair tresses as a result of the UV exposure was determined by analyzing the water extracts described above by MALDI-TOF analysis.
- Water extracts were mixed (1:1) directly with a MALDI matrix solution (5 mg of a-Cyano-4-hydroxycinnamic acid (CHCA) dissolved in 1 ml of 80:20:0.1 acetonitrile: water: trifluoroacetic acid). About 1 ul each sample was spotted on the MALDI plate and analyzed by MALDI-TOF/TOF 4800 plus system (AB-Sciex).
- a protein marker for hair damage from UV was detected in the water extracts with m/z 1278.
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US13/109,546 US20110281256A1 (en) | 2010-05-17 | 2011-05-17 | Systems and Methods of Detecting and Demonstrating Hair Damage Via Evaluation of Protein Fragments |
US14/217,977 US9310351B2 (en) | 2010-05-17 | 2014-03-18 | Systems and methods of detecting and demonstrating hair damage via evaluation of protein fragments |
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US34532110P | 2010-05-17 | 2010-05-17 | |
US35439710P | 2010-06-14 | 2010-06-14 | |
US38946910P | 2010-10-04 | 2010-10-04 | |
US13/109,546 US20110281256A1 (en) | 2010-05-17 | 2011-05-17 | Systems and Methods of Detecting and Demonstrating Hair Damage Via Evaluation of Protein Fragments |
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US (2) | US20110281256A1 (fr) |
EP (1) | EP2572201B1 (fr) |
JP (1) | JP5513679B2 (fr) |
CN (1) | CN102893160B (fr) |
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US20150364312A1 (en) * | 2014-06-12 | 2015-12-17 | The Procter & Gamble Company | Systems and Methods Of Detecting and Demonstrating Heat Damage to Hair Via Evaluation of Peptides |
US20150362507A1 (en) * | 2014-06-12 | 2015-12-17 | The Procter & Gamble Company | System and Methods of Detecting and Demonstrating Ultraviolet Damage to Hair Via Evaluation of Protein Fragments |
US9310351B2 (en) | 2010-05-17 | 2016-04-12 | The Procter & Gamble Company | Systems and methods of detecting and demonstrating hair damage via evaluation of protein fragments |
US9671410B2 (en) | 2011-01-16 | 2017-06-06 | The Procter & Gamble Company | Biomarker-based methods for identifying and formulating compositions that improve skin quality and reduce the visible signs of aging in skin |
US10966916B2 (en) | 2014-11-10 | 2021-04-06 | The Procter And Gamble Company | Personal care compositions |
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US11365397B2 (en) | 2018-11-29 | 2022-06-21 | The Procter & Gamble Company | Methods for screening personal care products |
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JP6097636B2 (ja) * | 2013-05-20 | 2017-03-15 | 一般社団法人日本真珠振興会 | 真珠核の識別方法 |
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- 2011-05-17 CN CN201180024698.5A patent/CN102893160B/zh active Active
- 2011-05-17 US US13/109,546 patent/US20110281256A1/en not_active Abandoned
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Also Published As
Publication number | Publication date |
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JP2013526712A (ja) | 2013-06-24 |
US20140197309A1 (en) | 2014-07-17 |
CA2798517C (fr) | 2016-07-05 |
EP2572201A1 (fr) | 2013-03-27 |
CN102893160A (zh) | 2013-01-23 |
CA2798517A1 (fr) | 2011-11-24 |
WO2011146462A1 (fr) | 2011-11-24 |
JP5513679B2 (ja) | 2014-06-04 |
EP2572201B1 (fr) | 2015-08-26 |
CN102893160B (zh) | 2015-10-14 |
US9310351B2 (en) | 2016-04-12 |
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