New technologies allow analysts to extract more information from DNA than ever before—but what can be done when nuclear DNA is not available or is too degraded to use? Researchers at Battelle are investigating new methods that use proteomics, or the analysis of proteins, to generate forensic data.
The proteome is the complete set of proteins that are produced or modified by an organism, system or cell. Studying the sequences of proteins in a sample can give us important clues about the DNA of the organism that produced them. Just as every individual has a unique genome that varies in small but important ways from others in their species, the proteome of each individual is unique. This means that it can potentially be used as an alternate method of identification in forensic investigations.
For example, hair is composed primarily of keratin, a fibrous protein that also comprises human fingernails and horns and hooves in animals. The cells contained in the hair shaft are dead and therefore do not contain nuclear DNA. That means that unless the sample contains the living cells at the root, hair samples do not provide nuclear genomic DNA for sequencing. However, proteomics may one day be able to provide an analysis of the protein sequences in the hair samples that could be used for human identification. It could also be useful in situations where DNA is too degraded to use, such as old blood or saliva stains. Proteins are often more stable than DNA molecules, so analysts may be able to recover usable information from proteins in samples of body fluids even if the DNA is not recoverable.
The science of forensic proteomics is still in its infancy, and much work remains to be done before proteomic data can be used for forensic purposes. In large part this is because proteomics is much more complicated than genomics. While each person has just one genome, each gene can produce multiple different protein variations from alternative splicing, post-translational modification and other events. However, forensic science can take advantage of these variations. Different cells in the body produce different proteins at different times as genes are switched on or off by body system activity or environmental factors. That means the proteins found in your saliva will look very different from the ones found in your blood, and the proteins found in a saliva sample after a heavy meal will be different from those found after a long fast. Thus, the proteome is rich with information regarding sample origin, exposure and other forensically valuable information.
In cases where proteins are to be used simply for human identification, researchers need to identify a set of biomarkers that are consistent over time for individuals and have enough variability between individuals to be useful for identification. Genetic polymorphisms (nearly identical genetic sequences) among individuals can be analyzed at the protein level as well, provided the genetic polymorphism encodes for a protein sequence change. However, unlike DNA, proteins cannot be copied and amplified for analysis; the proteins within the original sample are all that will be available for analysis. Also unlike DNA, proteins are not sequenced directly. Rather, they are analyzed using mass spectrometry in order to extrapolate their sequences. This makes analyzing and sequencing proteins more complicated and prone to interpretation errors than genomic sequencing.
Researchers at Battelle are investigating methodologies to make protein analysis easier, faster and more accurate. In order to make proteomic data useful for forensic purposes, the industry will need to develop standards and protocols for protein forensics, including extraction techniques, sample preparation protocols and analytical methods. Battelle’s proteomics work builds on similar work conducted over the last decade to advance the science of DNA forensics as well as proteomics for non-forensic applications. For the last few years, Battelle researchers have been heavily involved in developing, validating and evaluating new methods and technologies to bring Next Generation Sequencing (NGS) into forensic laboratories. Their proteomics research seeks to develop similar standards and methodologies for protein analysis. Specifically, they seek to define:
- Methods for sample preparation and protein extraction for various sample types
- Analytical methods to identify variations in the polypeptide sequences of proteins
- Statistical calculations and algorithms that can be used to identify the variations that have the most forensic meaning
If researchers can identify the useful variations in the polypeptide sequences between individuals, those variations could be used to develop a reference panel for forensic identification.