27 Proteomics, Genomics & Microarrays Figure 3
Figure 1 a
Non-productive MS2 scan region
Sequential quadrupole
isolation of fragment ions
MS2 Precursor ion isolation
Precursor ion fragmentation
Fragment ion spectra
b MS2 TIC ENDO Precursor ion isolation
Precursor ion fragmentation
Simultaneous fragment ion detection by Orbitrap analyser
Sequence ion MS/MS spectra
MS2 Quantification 19
SRM and PRM are conventional targeted LC-MS approaches. (a) In SRM assays the mass spectrometer is programmed to monitor for the presence of one or more precursor ions, collisionally fragment these ions, isolate, and detect the resulting fragment ions in sequential steps. The integration of extracted ion chromatograms (XIC) from the diagnostic fragment ions allows quantitation of the target. (b) PRM shares similarities with SRM, however, fragment ions are isolated and detected in parallel using high-resolution, accurate mass detectors such as Orbitrap mass analysers. This enables post-acquisition determination of the optimal fragment ions for quantifi cation as well as higher measurement selectivity.
SRM and PRM are conventional targeted LC-MS approaches. (a) In SRM assays the mass spectrometer is programmed to monitor for the presence of one or more precursor ions, collisionally fragment these ions, isolate, and detect the resulting fragment ions in sequential steps. The integration of extracted ion chromatograms (XIC) from the diagnostic fragment ions allows quantitation of the target. (b) PRM shares similarities with SRM, however, fragment ions are isolated and detected in parallel using high-resolution, accurate mass detectors such as Orbitrap mass analysers. This enables post-acquisition determination of the optimal fragment ions for quantification as well as higher measurement selectivity.
For both SRM and PRM, a major constraint lies in the relationship between target multiplexing (the number of analytes that can be measured reliably) in the desired cycle time and the amount of time devoted to each analyte within that timeframe. Cycle time is constrained by the properties of the LC setup, and so there is always a tradeoff between the highest performance (in terms of selectivity and sensitivity) and number of targets per analysis. Increasing the amount of time devoted to a single analyte brings improved sensitivity, for instance, but allows fewer analytes to be quantifi ed. Conversely, large numbers of targets cannot be studied for as long, compromising data quality. Overall, LC-MS approaches either bring high-scale coverage with sub-optimal quantitation, or low- scale coverage with high quantitation, but not both.
Figure 2 Sensitivity
Cycle time
Trapping ions Fill time
20 21 22 23 24 [min] 19 20 21 22 23 24 [min]
Fragment ion XIC
Quantification 19 20 21 22 PRM 23 24 [min] 19 LCDSGELVAIK MS2 TIC 20 21 22 SureQuant 23 24 [min] MS2 TIC IS Productive MS2 scan region MS2 TIC
SureQuant intelligent detection of targets maximises instrument efficiency and productivity. The IS and endogenous detection of a representative peptide, LCDSGELVAIK, is shown from PRM and SureQuant acquisition. In the PRM experiment, many uninformative MS2 scans are captured for the IS and endogenous target (grey region) during the 2.5 min monitoring window, and a smaller proportion of MS2 scans are captured during the actual target elution time (white region). The dynamic nature of SureQuant acquisition minimizes unproductive scans allowing shorter duty cycles and higher productivity. Experiment details: 50 fmol IS spiked into 250ng HeLa cell line digest. PRM MS2 settings: 2.5 min RT window, 15000 resolution, 20 ms IT. SureQuant MS2 settings: Watch mode 7500 resolution, 10 ms IT; Quant mode 60000 resolution, 116 ms IT.
SureQuant intelligent detection of targets maximises instrument effi ciency and productivity. The IS and endogenous detection of a representative peptide, LCDSGELVAIK, is shown from PRM and SureQuant acquisition. In the PRM experiment, many uninformative MS2 scans are captured for the IS and endogenous target (grey region) during the 2.5 min monitoring window, and a smaller proportion of MS2 scans are captured during the actual target elution time (white region). The dynamic nature of SureQuant acquisition minimizes unproductive scans allowing shorter duty cycles and higher productivity. Experiment details: 50 fmol IS spiked into 250 ng HeLa cell line digest. PRM MS2 settings: 2.5 min RT window, 15000 resolution, 20 ms IT. SureQuant MS2 settings: Watch mode 7500 resolution, 10 ms IT; Quant mode 60000 resolution, 116 ms IT.
Refi ned IS-PRM approaches such as SureQuant bring superior acquisition effi ciencies of 80-90% (cf. 10-15% via other conventional targeted approaches), as acquisition parameters can be adjusted on-the-fl y to maximise sensitivity and selectivity at the time- point when target is eluting. This productivity brings enhanced data quality and allows more targets to be quantifi ed in the same amount of analysis time to increase target scale and throughput. Importantly, it enhances the chance of successful detection, as the use of internal standards intelligently guides measurement of the target of interest at precisely the right time.
[s] 20-30 s Cycle time (2-3 s) Scale Target Multiplexing Number of peptides/cycle Selectivity
Background interference: HR/AM Resolution (~ transient time)
The chances of missing a target measurement are, therefore, greatly minimised, resulting in more consistent, reliable and robust measurement and quantitation. Obtaining a more reliable and detailed dataset allows researchers to make fully informed decisions, and can help to meet compliance needs in highly regulated environments by enabling more precise quantifi cation of the substances within a drug product.
Inter-dependencies between experiment scale, sensitivity, and selectivity. Chromatographic elution properties of the analyte and the desired sample rate (left) will ultimately determine the amount of time the mass spectrometer can spend taking measurements (centre). Within this fi xed time, the instrument can be used to collect fewer measurements with high selectivity and sensitivity or alternatively, take more measurements but a reduced sensitivity (right).
Inter-dependencies between experiment scale, sensitivity, and selectivity. Chromatographic elution properties of the analyte and the desired sample rate (left) will ultimately determine the amount of time the mass spectrometer can spend taking measurements (centre). Within this fixed time, the instrument can be used to collect fewer measurements with high selectivity and sensitivity or alternatively, take more measurements but a reduced sensitivity (right).
The way forward for proteomics researchers
The limitations of common LC-MS and antibody-based quantitative proteomics methodologies are far from abstract: they have real-world impacts on patients. They result in new therapeutic options either being delayed or unavailable, due to a limited understanding of how tumours and diseased cells signal, and how best to target them via treatment.
To overcome these hurdles, a new approach – internal standard triggered PRM (IS-PRM) – was developed that can dynamically guide targeted analysis in real time. This allows large numbers of targets to be reliably measured at high sensitivity and selectivity, protecting data quality and improving the effi ciency of the analytical process. However, early iterations of IS-PRM require an advanced level of technical knowledge, with the user needing to utilise complicated programming interfaces and informatics tools to develop assays and prepare methods that achieve only a partial implementation. This has limited the uptake of IS-PRM by the proteomics community.
More recent developments remove this barrier. The Thermo Scientifi c SureQuant IS Targeted Quantitation method, for instance, is an evolution of the original IS-PRM approach. It presents the methodology in a usable, easily implementable way as a turnkey ‘off the shelf’ solution for any laboratory, including for use in biotherapeutics, plasma analysis, host-cell protein analysis, and to replace traditional biochemical analytical approaches such as ELISA.
Targeted proteomics workfl ows have traditionally been challenging, especially for people new to the technology, as the amount of time and effort needed to develop, standardise and validate a targeted assay can be considerable. IS-PRM technologies, such as the SureQuant method, simplify this by bringing an intelligent approach to proteomics, which in turn maximises chances of successful detection and quantitation.
6 8
Original elution
window
Heavy Peptide
Offset elution
Detection Offset PRM " 16 18 RT (min) 20 22 SureQuant ! Figure 4
A
B A B
C C
D D
10 12 14 16 18 20 22 24 26 28 30 32 RT (min)
! Original !
Original elution profile
5 min offset elution profile
SureQuant acquisition robustness overcomes chromatographic fluctuations. Targeted analysis of AKT-mTOR pathway proteins was performed by PRM and SureQuant acquisition using a standard gradient and an offset gradient which introduced a 5 minute artificial time delay to simulate LC retention time variations that can commonly occur (landmark peptides A-D are indicated for comparison). As an example, the observed heavy peptide LFDAPEAPLPSR (m/z 611.8526 ++) elution is shown at the original and offset retention times (bottom left). Notably, while PRM acquisition failed to capture the signal of the peptides with delayed elution, SureQuant acquisition maintained reliable measurement under these conditions.
SureQuant acquisition robustness overcomes chromatographic fl uctuations. Targeted analysis of AKT-mTOR pathway proteins was performed by PRM and SureQuant acquisition using a standard gradient and an offset gradient which introduced a 5 minute artifi cial time delay to simulate LC retention time variations that can commonly occur (landmark peptides A-D are indicated for comparison). As an example, the observed heavy peptide LFDAPEAPLPSR (m/z 611.8526 ++) elution is shown at the original and offset retention times (bottom left). Notably, while PRM acquisition failed to capture the signal of the peptides with delayed elution, SureQuant acquisition maintained reliable measurement under these conditions.
Signal
[m/z]
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