Keratin contamination

One should be aware of peptide contaminates observed in the spectrnm. One s sample may be a mixture of proteins that will resnlt in a mixtnre of unrelated tryptic peptides. It is also essential to prepare the sample in a clean environment. Otherwise, keratin contamination may be observed in the spectrum. Unused pipet tips should be covered and wool clothing shonld not be worn in the laboratory. In addition, trypsin will undergo antolysis and these tryptic peptides will often be observed in the mass spectrnm. 

These authors also mention some shortcomings that should be borne in mind, in particular, that some peptides observed were from the autolysis of trypsin, the digestion agent, and from contaminants such as human keratin, while some peptide ions did not produce interpretable MS-MS spectra. 

The easiest way to detect a protein modification seems to be the mass measurement of all peptides generated by enzymatic digestion. The comparison with the predicted peptide masses from the sequence of the protein identifies unmodified peptides and unexplained masses would give indications to modified peptides. Unfortunately, this is not a suitable approach in practice. In many peptide mapping experiments done with the MALDI mass mapping technique, up to 30% of the measured masses remain unexplained. This is probably due to protein contaminations from human keratins, chemical modifications introduced by gel electrophoresis and the digestion procedure, and other proteins present at low levels in the piece excised from the sodium dodecyl sulfate gel. The detection of a protein modification requires a more specific analysis. 

The proteins are then transferred from the gel to nitrocellulose (Methods in Molecular Biology, Volume 10, Chapters 24-26 and ref. 5). It is important to work with gloves when handling nitrocellulose to avoid contamination with skin keratins. 

More than one protein present in the sample with good-quality PMF data, mixtures containing two to three proteins can be identified, but the presence of peptide masses from the other proteins reduces the score for each individual match. With lower-quality data, this can result in all of the scores dropping below the significance threshold. The presence of contaminants, e.g., trypsin or keratins, has the same effect however, known peptide masses derived from keratins and trypsin can be removed from the PMF data prior to searching (9). 

Hair serves to eliminate toxic materials (e.g., lead) and metabolites from the body, and may be used to monitor environmental contamination. For example, copper deficiency is a cause of Menke s kinky hair syndrome protein deficiency leads to hair loss and discoloration. Hair keratin carries a strong negative charge and binds inorganic materials it becomes prone to... 

Several in vivo and in vitro studies conclusively demonstrate that benzene can be absorbed through human skin (Blank and McAuliffe 1985 Franz 1984 Susten et al. 1985 Tsuruta 1989). In general, skin absorption is considered a minor source of concern in the occupational environment as it occurs at a much lower rate and extent compared with benzene absorption through the respiratory system (OSHA 1989). However, benzene absorption through the skin as a result of benzene contamination in rubber solvents is a major route of exposure in tire building operations (Susten et al. 1985). Benzene is an irritant to the skin and, by defatting the keratin layer, may cause erythema, vesiculation, and dry and scaly dermatitis... 

One of the most significant problems with this work is contamination of the samples with environmental proteins (such as keratin from skin and hair cells). Therefore, care should be taken to work with clean materials and, if possible, operate in positive pressure hoods to decrease the potential for contamination. Also, the Eppendorf-type tubes used in these procedures should be rinsed in 0.1% formic acid dissolved in 50 50 water acetonitrile, dried under vacuum, and stored under dust-free conditions. 

Some of the precautions that can be exercised should be a normal part of good laboratory practice, and include the appropriate use of personal protection. Covering the hair and using gloves will minimize the possibility of contamination from skin and hair-derived keratin proteins, as well as amino acids, fatty acids, and cosmetic residues from the skin surface. 

It should be noted that during the preparation of any protein or peptide sample, some of the analyte will be permanently adsorbed to the walls of the vial, to packing material, or to any transfer device used such as a pipet tip. The loss of sample is of critical concern when working below approximately 100 fmole/pl. Protein adsorption by some vials is so great that the sample will not be observed in the mass spectrum. In addition, contamination from airborne protein particles, such as human or sheep keratin, can cause misleading results when working at these low levels. Covering your pipet tips and vials is recommended for protein identification work. 

Step 2. Purify the protein in a system in which the modification is stable. Avoid any kind of external contamination during purification, for example, keratin, which is the most common impurity present in MS analysis. If purification is not possible, it can be enriched, separated, and recovered from an SDS-PAGE. The amount of sample is also important. A very sensitive machine can identify the protein at the famtomole level. 

Chemical contaminants may bind to and react with keratin proteins in the stratum comeum of the skin in occupational exposures under field conditions. The tapestripping method was successfully applied for the removal and quantification of keratin from the stratum comeum for normahzation of extracted amoimts of naphthalene (marker hydrocarbon of jet fuel) from human volimteers experimentally exposed to JP-8 jet fuel (Chao and Nylander-French, 2004). Another study indicated that the naphthalene has a short retention time in the human stratum comeum, and that the tape-stripping method, if used within 20 min of the initial exposure, can be used to determine the amoimt of naphthalene initially in the stratum comeum following a single jet fuel exposure (Mattorano et al., 2004). These studies emphasized... 

When analyzing peptides by LC-MS, especially with nano-LC, several of the contaminants listed earlier (Section 3.2.6) are encountered, particularly those that are PEG-based and various siliconates. There are also contaminants specific to peptide analysis, such as the products of autolysis, that occur when too much trypsin is added (or when the sample contains less protein than expected) (Table 3.9a). As a rule of thumb, trypsin should be present at a concentration that is 1% of the protein to be digested. Another common contaminant is keratin that is introduced by incorrect handling of samples or by accidental contamination during sample collection (Table 3.9b). 

An outbreak of chloracne in 17 workers from a British plant manufacturing dichloro-aniline-derived herbicides was reported in 1993. TCAB and TCAOB were the acnegens. Comedones evolved 6-12 weeks after exposure to these chloracnegenic contaminants. Cutaneous xerosis and folliculitis, on the trunk, limbs, thighs and buttocks, previously uncommonly described, was present in 50% of exposed workers. Affected follicles were surrounded by a collarette of scale and frequently the hair shaft was twisted or broken. The pathogenesis of these lesions is unclear but may involve a disorder of keratinization. A direct toxic effect on epidermal keratinocytes or a secondary effect due to a perifollicular inflammatory reaction has been theorized (McDonough et al. 1993). They suggested that folliculitis and xerosis should be included in the clinical spectrum of chloracne. 

Artifactual bands with molecular weights ranging from 50,000 to 68,000 are often observed in silver-stained gels. Evidence has been presented indicating that these bands are due to contamination by keratin-type proteins (Ochs, 1983). These bands may be more prominent in the presence of certain reducing agents, such as mercaptoethanol. 

While the essentially keratinous bulk makeup of hair and skin (SC) is generally accepted, their surfaces are less well defined. It is obvious that the detailed properties of the latter will be pivotal in determining the adsorption behavior of added conditioning agents. Thus, it is recognized that a sebum layer is generally/Irequently present on both of these substrates (see below). While such a layer can be regarded as a contaminant, a lipid layer can also be intrinsic to the surface. This aspect has been explored in some detail in the case of hair (2), the substrate of emphasis in this chapter. For details on the structure of hair the book by Robbins (2), and Chapter 12, may be consulted. 

---