Independence Encouraged, Supervision Not Required
Established in 1993, Kansas State University's Biotechnology/ Proteomics Core Lab is a facility that provides services to researchers at the university and other research and development (R&D) institutions. The lab assists scientists with projects related to new protein identification, protein modifications, protein-protein interactions and enzyme substrates.
Because of the agricultural emphasis at Kansas State University, many of our projects center on arthropods and plants, says John M. Tomich, director of the Biotechnology/Proteomics Core Lab. Some of the systems we work on include various strains of bacteria; the red flour beetle, tobacco horn worm and yellow meal worm; crickets, aphids, mosquitoes, ticks and thrips; wheat, soy beans and rice; and Arabidopsis.
These projects involve processes such as annotating the genomeslooking at where and when certain proteins are expressed developmentally or in which tissueor identifying enzymes involved in an insects biological processes such as digestive systems, immune systems, exoskeleton development and hormone signaling.
In plants, we have identified proteins that are affected by various growing conditions, insect or microbial attack, and storage conditions, explains Tomich.
The lab also synthesizes peptides, chromophoric enzyme substrates, fragments for antibody production, antigens and flavor enhancers and acts as a brokerage for buying and selling oligonucleotides, or polymers with a short sequence of nucleotides.
This lab has been in existence for 18 years and has dramatically changed during that period, says Tomich, who helped found the lab and has been heading it since. Initially we carried out oligonucleotide synthesis, amino acid analysis, automated Edman degradation and peptide synthesis. Of those tasks, only peptide synthesis remains. In 2005, we received money from the National Science Foundation (NSF) to equip the proteomics lab.
As director, Tomich oversees the entire facility and is in charge of the peptide synthesis and protein characterization elements. Working with him is Associate Director Yasuaki Hiromasa, who, with the help of an assistant, takes care of the proteomics section.
My background is in bio-organic (peptide chemistry) and bio-analytic chemistry, while the associate directors training is in enzymology and his assistants training is in biomedical engineering, Tomich says.
Together, they run the core lab, which occupies approximately 1,500 square feet of space in two separate rooms, and two offices. “One room houses the mass spectrometers and high-performance liquid chromatography (HPLC) systems, while the second room houses our electrophoresis and robotics units for picking spots,” says Tomich.
Within this facility, Tomich and his staff assist clients who are mostly within Kansas State University and the nearby United States Department of Agriculture (USDA) Agricultural Research Service (ARS) facilities. The lab does, however, take samples from other institutions and industries.
“In 2009, we had 93 projects [that] used the oligonucleotide service, 40 projects that used the mass spectroscopy service, 12 projects that used the peptide/protein synthesis/separation service and 10 other projects that used other services provided by the core lab,” explains Tomich.
In the areas of plant and arthropod biochemistry, proteomic studies are at the forefront of research. The proteomics work that Tomich and his staff conduct in these areas has helped a number of their clients receive federal grants for their projects.
“It has given us great satisfaction to see our clients receive new funding and international acclaim for these projects,” he says.
Tomich and his staff receive orders daily. The oligo orders are processed through Integrated DNA Technologies, which sends samples back within 48 hours. By centralizing orders, the lab can provide competitive prices and free shipping. Orders involving peptides usually take several weeks to fill, while the proteomics work is usually turned around within 10 days.
One reason that operations run so smoothly is because Tomich relies on his competent staff—he says they do not require much supervision.
“I give my people lots of independence and let them know that it is the completion of projects that matters more than them punching a clock,” he says. “Flextime is the buzzword here. Folks come and go to make sure that everything is moving forward at a reasonable pace.
“I give the two staff people total independence to schedule the projects and maintenance of the instruments. I even give them time to establish independent collaborations and publish independently.”
Equipped with $3 million worth of scientific instruments, Biotechnology/ Proteomics Core Lab is not just used for providing services to plant and animal researchers on campus and in industry, but is also used as a teaching tool for students in the field.
“We like to engage students and have them work with our instruments so that they will have a better idea of where problems arise, as well as have a working knowledge so that when they present their data they know what they are talking about,” Tomich says.
For example, two graduate students— both with chemistry backgrounds and nearing the completion of their degrees— help with peptide synthesis projects.
Not only does their work help the lab, but both of these students use peptide synthesis in their own research as well, Tomich explains. This type of involvement allows the students to be part of important research and have a chance at having their work published.
“With the exception of the nano-LC MS/MS, proteomics students and faculty researchers can train on our instruments and [also] sign up to use them as time permits,” Tomich says.
Through support from Kansas University’s upper administration, Tomich and his staff have been able to offer their proteomic services at a greatly reduced cost to junior investigators and those seeking to change the focus of their projects.
“All we charge are replacement costs for the consumables,” Tomich says. “Through this mechanism, we have helped a number of junior investigators get extramural funding and advance in their careers.”
The lab is also used for Tomich’s own federally funded research. “We have three different projects that look at bioactive peptides that function either as drugs or help deliver drugs. My research uses most of the instrumentation housed in the core lab.”
Inventory, maintenance, hiring
The core lab is divided into proteomics, peptide synthesis and protein characterization.
Within the area of proteomics, the lab staff primarily uses two different types of mass spectrometers to analyze protein samples partially separated using 1d and 2d gel electrophoresis. Specifically, Tomich and his staff use a Bruker Ultraflex III MALDI TOF/TOF—an instrument for accurate mass determination that requires miniscule amounts of the sample and is capable of MS/MS analyses of certain high-abundance peaks. The team specifically uses this machine for peptide mass fingerprinting (PMF) and in-sourcedecay (ISD) analyses to identify proteins and some post-translational modifications (PTMs).
“Our other instrument is a Bruker HCT-Plus, which we use for nanoflow LS MS/MS analyses on in-gel digested proteins that are cut out of gels,” says Tomich of the instrument they routinely use to generate peptide sequences. “All of the workflow is processed by the Bruker program, Proteinscape, with the aid of the search engines Mascot and Phenyx.”
“For peptide synthesis we are set up to perform either FMOC or t-BOC chemistries. We prepare full-length bioactive compounds, chromophoric enzyme substrates, fragments for antibody production, antigens and flavor enhancers,” Tomich adds. They can be linear or cyclic and can contain heavy atom labels, dyes, biotin, cholesterol and other specific molecules.
“We automated Applied Biosystems Inc. peptide synthesizers that run our proprietary synthesis programs.”
For protein characterization, the team has a number of instruments with various uses, including circular dichroism (CD), Fourier transform infrared (FT-IR), fluorescence, plasmon resonance and analytic ultracentrifugation to look at either protein secondary structure or protein-protein interactions.
In order to ensure that all these processes run smoothly, the team has to stay on top of inventory and maintenance. The associate director, Hiromasa, maintains the inventory and ensures that the lab is well stocked for smooth operation. Because the lab is situated in a location that isn’t in the vicinity of many service companies, Tomich and his staff maintain most of the instruments themselves.
“This past year we purchased a oneyear service contract on the two MS instruments to have them repaired and upgraded,” Tomich says. “For our older peptide synthesizers, I am always on the lookout for old units that can be used for parts.”
Tomich also takes care of the hiring. Although he says that in the 18 years of the lab’s existence, he’s seen minimal turnover.
“The former associate director took a tenured faculty position in Japan and two assistants have moved on to other positions for family reasons.”
Challenges and rewards
Tomich finds that a big challenge is to constantly remain relevant to the research interests of his client base. Because of this, the lab staff is continually modifying services to meet industry needs and actively seeking funding to keep up with the field.
“This is why we dropped certain analyses and added others,” Tomich says. “We are just beginning to get requests for post-translational modification analyses and will need to get new instrumentation to fulfill these requests.”
In addition to the physical tools that the lab seeks, Tomich employs another valuable tool— communication.
“Communication is very important; making sure that the staff understands exactly what the clients are asking for is critical,” he says. “We insist that all requests be in writing.”
Another way to ensure that the client’s needs are being addressed is to meet with all the clients on a regular basis— to go over their needs, procedures and schedule of charges. Tomich also sends his clients any updates that might aid their research.
“Additionally, it is important that the clients know both our strengths and limitations. If they want something that we cannot do, I refer them to other labs that can provide the service.”
For Tomich, being the director of Kansas State University’s Biotechnology/ Proteomics Core Lab is rewarding because in addition to conducting his own internationally recognized projects, he gets to participate in the projects of other researchers.
“These can be both mentally stimulating and challenging,” he says. “No two days are ever the same. Every day brings about some new discovery or problem to overcome.”
But it’s the overall contribution of the lab to society and science that makes the hard days and challenges worth it.
“Many of the projects that we are working on have implications in pest management and crop plant nutritional value,” Tomich explains. An example is clients that seek to find species-specific enzyme targets for a new generation of environmentally safe pesticides.
Sara Goudarzi is a freelance writer based in New York City. Her Web site is www. saragoudarzi.com.
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