August 2021: Brown Lab
What is the main focus of your lab?
Quantitative Proteomics and Metabolomics Center
The focus of the center is the identification of proteins and metabolites with differential quantitative expression in cells, tissues or in protein affinity purifications, and we have applied this technique employing mass spectrometry to many different research problems with a strong focus on clinical, translational applications and basic science. A central focus is on stem cell science.
How long have you had your lab? When did you join Columbia University?
Where is your lab located?
Room 740 Mudd building, Morningside campus
What are the most exciting projects/directions in the lab at this moment?
In an early stem-cell related project, we used label-free protein profiling to study adipose-derived human stem cells under hyperosmotic treatment. In a related stem cell project, growth factor priming differentially modulated components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells. Another study used induced pluripotent stem cell-derived retinal pigment epithelium from patients provided age-related macular degeneration patient-specific cell lines. These allowed us to conduct an unbiased proteome screen to identify a genetic allele’s susceptibility of AMD. In a related project, we showed that HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1 in iPSC-derived RPE cell lines. Yet another stem cell effort focused on the influence of hypoxia and IFN-gamma on the proteome and metabolome of therapeutic mesenchymal stem cells. A related project demonstrated that dual IFN-gamma/hypoxia priming enhanced immunosuppression of mesenchymal stromal cells through regulatory proteins and metabolic mechanisms. Finally, a study on human brain tissue established hippocampal proteome biosignatures of major depressive disorder.
We are also conducting a large proteomics study of cerebrospinal fluid in familial and sporadic Alzheimer's disease patients. In other projects, emphasis is on large-scale quantitative protein profiling as well as the study of posttranslational modifications, particularly phosphorylation leading to the understanding of novel cell death mechanisms in cancer cells. Other diverse collaborations deal with diet and bariatric surgery, liquid phase transitions as organizers of chromatin structure, changes in the proteome of the human cervix during pregnancy and the impact of implantation of left ventricular assist device on the human aortic valve proteome. In our collaborative laboratory (http://proteomics.bio.columbia.edu) we continue to participate in intensive, long-term and large-scale collaborations. Our role includes project planning, experimental design, sample processing, peptide enrichments, LC/MS, computational and pathway analysis, data interpretation and authorship of publications. We emphasize bottom-up proteomics with a Q Exactive HF Orbitrap from Thermo Scientific.
What are the biggest accomplishments that your lab recently had?
Numerous published papers demonstrate our focus on using proteomics and mass spectrometry to study stem cells. One project with cancer stem impact is regulation of ferroptotic cancer cell death by glutathione peroxidase 4 (GPX4). Using mass spectrometry-based proteomics in our lab, we were the first to describe the central role of GPX4 in ferroptosis, a non-apoptotic cell death mechanism demonstrated to be important in cancer stem cells. This work (Yang et al. Cell. 2014, 156:317-331) was highly cited (>1,000 citations) and had broad impact. GPX4 maintains a stemness phenotype, oxidative homeostasis and regulates biological processes in cancer stem‑like cells. Our work stimulated a broad range of follow-on studies from many groups including demonstrating that small-molecule ferroptotic agents have potential to selectively target cancer stem cells. “Persister” or drug-tolerant cells have been described as cells surviving cytotoxic drug exposure. Recent studies with various cancers have indicated that the molecular signature of “persister” cells includes upregulation of stemness factors and GPX4 and results in abrogation of ferroptosis, allowing cells to survive after cytotoxic drug exposure during chemotherapy.
What are the key techniques that your lab is using?
Shotgun proteomics is a strategy with broad applicability using liquid chromatography-mass spectrometry (LC/MS). These robust and flexible platforms allow the design of large analyses in clinical patient samples, cell culture or animal model projects.
What facilities or equipment does your absolutely lab rely upon? Do you use CSCI cores?
In a typical experiment, proteins are extracted and digested with proteolytic enzymes. Following reduction, alkylation of cysteines, peptides are separated on an UltiMate 3000 RSLCNano liquid chromatograph (LC) coupled to a Q Exactive HF Orbitrap mass spectrometer. This system has been demonstrated to exhibit exceptional performance by other groups and in our own published work.
We use Peaks Studio (Bioinformatics Solutions, Inc.) software used for database searching and quantitative analysis. This software extends the dynamic range of fold-change data, especially for low abundance proteins. This uniquely powerful system matches accurate mass and retention time of peptides across all liquid chromatography/mass spectrometry (LC/MS/MS) chromatograms in an experiment, multiplying the effectiveness of our leading edge mass spectrometry platform. The software includes statistical tools and false discovery rate corrections.
Who shall be contacted with questions about equipment, resources and training?
What's your best approach to mentoring trainees in the lab?
We provide training in all aspects of instrument operation and maintenance, data collection, processing and interpretation. This equipment and data-analysis pipeline requires intensive day-to-day attention. We use collaboration onsite and even web-based remote access tools to interact as a team to manage these complex instruments and analysis pipelines. While this is a team effort, trainees take center stage to receive credit for any accomplishments. We provide unwavering support for career development.
Can you recommend courses/lectures in Columbia University that would be most beneficial for students/postdocs?
I teach a laboratory course every fall semester (Proteomics Laboratory, Biology 4260, 3 credits). It is open to graduate students, postdocs and advanced undergraduates. We have even had faculty who are contemplating projects attend. Lecture/laboratory topics include introduction to mass spectrometry, hands-on sample preparation, LC/MS access in supervised hands-on tutorial sessions, and extensive training in database searching data analysis and interpretation.
What would be your career advice for students/postdocs?
You need to work in an energetic collaborative group that has a good track record. However, in some fields there are limited career opportunities, regardless of the lab or how hard the trainee works. In our field, mass spectrometry-based proteomics, there is an extreme shortage of trained scientists with the specialized skill set that we provide. All of the postdocs coming out of our lab have been aggressively recruited by the pharmaceutical industry and go directly into rewarding research staff scientist or senior research scientist positions. I cannot give advice to those in other fields.
Are you accepting rotating students at the moment?
We do not sponsor students directly, but can co-supervise students depending on interest.
How do members of your lab celebrate accomplishments?
We generally have a celebratory luncheon at a restaurant, typically with champagne.
What is the key to running a successful lab?
Doing interesting work and respecting staff and their essential contributions.
What was the most exciting part about starting your new lab?
Building a new resource that would substantially benefit faculty staff and students at Columbia.
Stem Cell Directions:
Which stem cell conferences does your lab attend?
We have attended the annual NYSTEM conference (before NYSTEM was dissolved) and look forward to CSCI conferences.
What was the main reason of you joining CSCI? What are the beneficial aspects of CSCI membership for your lab?
Our focus is on stem cell biology and we continue to have many collaborations in that area, especially with CSCI member labs. Most of our equipment was funded by the New York State Stem Cell Science Board (NYSTEM) with matching funds CSCI and from other units within Columbia University.
What do you plan to bring to the CSCI community?
Proven, powerful tools as described above.