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EDUCATION :: MASS SPECTROMETRY continued from page 32


verification-stage studies, more flexible and scalable tech- niques are necessary. Label-free data dependent acquisi- tion (DDA) methods have proven particularly useful at meeting this need and can be used to directly compare relative abundances of proteins across multiple liquid chromatography (LC)-MS/MS experiments without the use of isotopic tags. These methods are generally used for genome-wide protein identification studies and are very effective at extending proteome coverage while minimiz- ing redundant peptide precursor selection. The primary advantage of this approach is that the number of sample comparisons is not limited, creating a comprehensive and scalable workflow.


Label-free protein quantitation is based on tandem MS analysis of the most abundant precursor ions. In contrast to stable isotope-labeling approaches, where differentially labeled proteins are combined and analyzed together, proteins studied using label-free approaches are measured individually. While this allows for comparison of multiple samples, any deviations arising from sample preparation or instrument use generate greater variability and reduce precision. Thus, DDA-based experiments require more repeat measurements to achieve statistical significance.4 The latest DDA workflow optimizations, including improvements in separation, acquisition, and data analy- sis, overcome the challenges around method standard- ization and experimental reproducibility. Improvements in the sensitivity of capillary flow high-performance LC (HPLC) technologies, for example, are enabling better separation of peptides, and ultimately, deliver more pre- cise data. Data can be further enhanced using the latest LC columns that are designed to achieve more consistent chromatographic separation by reducing mobile phase dead volumes.


Modern high-resolution accurate mass (HRAM) tech- nologies are also leading to improved reproducibility in biomarker verification. The latest generation of Orbitrap mass spectrometers, for example, provide increased acquisition speed and advanced peak determination to expand the number of peptides sampled, thus increas- ing peptide identification across varying data acquisition modes. Significant improvements in sampling depth, sequencing speed, and protein identifications provide better and more consistent data for enhanced run-to-run reproducibility and confident biomarker verification.


High-resolution verification workflows While DDA workflows are very useful for scalable bio- marker verification, achieving the required analytical sen- sitivity can sometimes be challenging. Data-independent acquisition (DIA) is an alternative label-free biomarker verification approach that overcomes this challenge. In a DIA analysis, a set of precursor acquisition windows are used to cover a broad mass-to-charge (m/z) range. All peptides within the defined m/z window are fragmented and a product ion spectrum for each detectable peptide is generated, providing multiplexed proteome-wide quantification of even low-level proteins. While DIA workflows are well suited for biomarker candidate analysis in human samples, challenges with analytical selectivity and dynamic range have led to the search for method improvements. The co-isolates and co- fragments that are sampled by broad acquisition ranges can produce highly complex MS/MS spectra, making


36 MARCH 2019 MLO-ONLINE.COM


confident analysis more problematic. This can be particu- larly challenging when working with clinical samples such as plasma due to the natural abundance and diversity of peptides and plasma proteins.


High-resolution DIA (HR-DIA) workflows based on hybrid quadrupole-Orbitrap MS technologies are helping to obtain more confident measurements from large-scale proteomics studies. HR-DIA workflows address challenges with sample complexity by using much narrower acqui- sition windows, an optimization that is possible by the increased mass resolution of modern Orbitrap instru- ments. With a greater ability for deconvolution of com- plex spectra, driving improved precursor selectivity and unbiased analysis, HR-DIA workflows increase fidelity and identification range, leading to more reproducible and comprehensive protein profiling.


Validating biomarkers with sensitive and specific protein quantification


Biomarker validation requires workflows with more directed analysis, capable of sensitive and specific protein quantification. While MS approaches for biomarker vali- dation have traditionally relied on selected-reaction mon- itoring (SRM) techniques performed using triple quadru- pole mass spectrometers, variation in the intensities of the product ions generated from the precursor ions can result in sensitivity issues. Parallel-reaction monitoring (PRM) is an alternative approach that uses hybrid triple quadrupole-Orbitrap technologies to achieve more sensi- tive protein quantitation by identifying the most intense product ions to analyze. PRM offers higher selectivity and high-throughput protein quantitation, ensuring confident peptide quantification.


Once biomarker selection is narrowed to a small num- ber of target peptides, PRM for targeted MS quantification allows the full MS/MS spectra to be acquired for each precursor. This enables higher analyte selectivity to be achieved than with SRM, facilitating better discrimina- tion of target peptides from co-eluting interferences pres- ent in complex biological matrices. High-resolution MS can also support PRM analysis, enabling detection of low abundance peptides common in biological samples and outperforming alternative methods in terms of absolute quantification.


Despite these advantages, standard PRM methods can be limited by inconsistent retention times. Tempera- ture fluctuations, inefficient mobile phase mixing, flow rate instability, or column contamination issues can all influence analyte retention times and ultimately affect method reliability. Direct retention time PRM (dRT-PRM) is an improvement to PRM workflows that can help to correct for these issues by monitoring and adjusting reten- tion time windows in real-time using internal standards. In addition, dRT-PRM offers further benefits in terms of the quality and precision of peptide measurements to improve analytical reproducibility.


Accelerating biomarker development with the Cancer Moonshot


Many of the greatest challenges associated with transla- tional proteomics workflows relate to lab-to-lab reproduc- ibility, method standardization, and scalability. These issues are well-recognized, and large-scale collaborative programs such as the Cancer Moonshot initiative are


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