SWATH® Acquisition is an innovative strategy for acquiring data on a TripleTOF® mass spectrometer. In a previous blog, we learned how SWATH works. Now let’s learn what it can do for different applications:
The list of applications and publications that use SWATH is extensive and continues to grow every month. A quick search on PubMed gives a sense of the breadth and promise of this exciting MS technique as it continues to develop and make inroads into new areas of research.
For more information, watch this 5 minute video MS/MS ALL with SWATH Acquisition >
SWATH was first described in 20121 as a new quantitative acquisition strategy for proteomics research. Because SWATH is so comprehensive and reproducible, the technique proved to be ideal for studying extremely complex samples that could contain 1000’s of analytes over a wide dynamic range – like in a proteomics sample. Often proteomics samples will be cell or tissue lysates where detection and quantitation of the low-level analytes in a reproducible fashion is necessary for making new discoveries and supporting scientific hypotheses. With SWATH, 1000s of proteins can be routinely quantified per run, reproducibly, and with high data completeness and quantitative accuracy. SWATH has been used for proteome-wide quantitation of proteins within specific organisms, investigating biological variations of enzymes, and quantitative phosphoproteomics, to name just a few. SWATH has been applied to discovery proteomics – because of its strengths in providing breadth and depth of coverage – as well as translational proteomics – because of its strengths in higher throughput and accurate quantitation. Indeed, SWATH acquisition continues to be a key strategy for proteomics researchers around the world.
Watch this Webinar on SWATH for Biopsy Samples >
Host-Cell Protein Analysis of Biologics
But proteomics isn’t the only application that can take advantage of the benefits of SWATH acquisition. Capitalizing on the power of SWATH for protein analysis, the Biopharmaceutical community took note and began to apply the strategy to the study of protein-based therapeutics, or biologics. When biologics are made, they are actually synthesized within host cells such as E.coli using the host cell’s own machinery to actually manufacture the biologic. During the process of purification, host cell proteins, or HCPs can be co-purified with the biologic. These HCPs can interfere with the efficacy and safety of the biologic and so it is important to determine the identities and amounts of any HCPs during the purification. Traditionally this has been accomplished using ELISA techniques, but SWATH proves to be better, faster, and more economical overall. Because SWATH is unbiased, the full HCP complement can be detected at sub-ppm levels in 1 hour. In fact, protein contaminants from any source can be detected, not just HCPs. Additionally, sequence variants and low-level modifications to the biologic can also be captured in the same data set or in experiments specifically designed for peptide mapping of the biologic. SWATH has also been used with CESI-MS for detection of HCPs from limited sample quantities in the early development phase.
Metabolite ID of Small Molecules and ADC’s
Small molecules can also be analyzed using SWATH. In the world of drug development, SWATH has proven to be a powerful strategy for identifying and quantifying drugs and their metabolites. This information is essential for interpreting pharmacology, pharmacokinetic, and toxicology data and is mandated by regulatory agencies. Because of its unbiased approach, SWATH acquisition can provide broad coverage for metabolite ID, capturing both predicted and unpredicted metabolites/catabolites. For example, SWATH can be used to streamline discovery microsomal clearance and metabolite ID. Here, it enables the identification of compounds with the greatest risk for high clearance (quantitative), while also identifying the softest spots for metabolism (qualitative), thus allowing acceleration of early drug discovery. The data can be collected in a high-throughput environment and minimizes incomplete metabolite information which can lead to repeated sample analysis and decreased productivity. When SWATH is combined with a sample preparation workflow that utilizes HepatoPac co-cultured hepatocytes for interspecies drug incubations, a one-stop solution is created for truly comprehensive identification and quantitation across multiple species in a single experiment. Data can be collected from drugs with highly varied structures and biotransformation pathways all in a relevant pharmacokinetic timeframe.
Antibody-drug conjugates, or ADCs, present a particular challenge for pharmaceutical companies. The usual small molecule metabolism of the payload molecule must be studied, in addition to the antibody itself and the ADC as a whole. The number of possible species that can be generated when considering the metabolic/catabolic fate of an ADC is vast. One must consider payload-linker species with one or more amino acids attached plus potential biotransformations of the linker and/or payload, plus any cleavage or degradation products. SWATH acquisition effectively addresses the complex task of performing metabolite and catabolite ID on ADCs by searching for all components of the ADC: payload, linker and antibody, plus cleavage metabolites and biotransformations, thereby ensuring success for scientists studying ADC metabolism.
Metabolomics and Lipidomics
In the field of metabolomics and lipidomics, SWATH has also proven to be very useful. In a webinar from Professor Oliver Fiehn from the University of California Davis, Dr Fiehn highlights how SWATH combined with his in-house developed MS DIAL software provides improved analyte coverage over traditional information-dependent analysis (DDA) experiments for metabolomics, highlighting an increase of 66% in identified algae lipids. In another webinar featuring Professor Jeffrey McDonald from UT Southwestern Medical Center, SWATH Acquisition for infusion based lipidomics enabled very good to excellent coverage of most lipid classes with throughput on the order of 100 samples per day.
Forensic Drug Screening
But besides omics applications and applications within the pharmaceutical industry, SWATH Acquisition has also been used for forensic drug screening. For screening applications, often there is no prior knowledge about the numbers and identities of the drugs in the samples, but there is a need to report all correct identifications (true positives) and not to report erroneous compounds (false positives) or miss correct compounds (false negatives). Traditionally IDA approaches have been used, but because intense ions take higher priority within any data cycle, less abundant species can be missed in complex sample matrices when using IDA. SWATH Acquisition proves to be a viable tool for screening applications in both human urine and blood samples with >90% positive detection rate in blood samples and 95% detection rate in urine samples. Additionally, SWATH enables more sensitive detection of lower concentration species vs. traditional IDA approaches, and is compatible with ultra-fast screening. SWATH is also useful for screening human urine for bath salts. Bath Salts refer to a group of drugs containing one or more synthetic chemicals related to cathinone, one of the psychoactive principles naturally found in khat (Catha edulis Forsk) – an amphetamine-like stimulant. Because new synthetic cathinones are constantly emerging from underground chemists, the ability to retrospectively analyze the data from a previous sample for new drugs makes SWATH particularly useful in the fight to stay abreast of this major public health threat.
How about food? What could be more important than ensuring the integrity of the food we eat? SWATH acquisition is also extremely useful for screening foods for pesticides. New pesticides are constantly being synthesized, and with SWATH these new pesticides can be analyzed without creating new assays or purchasing new standards. A generic SWATH acquisition method enables analysis of all the components in a given sample, whether known or unknown. For example, food from a local grocery was screened using SWATH to determine differences between organic and non-organic vegetables. Multiple pesticides were identified from the non-organic sample. The data-independent nature of SWATH acquisition makes re-analysis without re-acquisition possible. With SWATH’s unbiased, non-targeted acquisition, new compounds or regulations are easily dealt with using the current SWATH strategy.
Watch this Webinar on SWATH for Screening Baby Food >
1.Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis. Ludovic C. Gillet,Pedro Navarro,Stephen Tate,Hannes Röst,Nathalie Selevsek,Lukas Reiter, Ron Bonner,and Ruedi Aebersold, Mol Cell Proteomics. 2012 Jun; 11(6). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433915/