The era of 4D-Omics
Mass spectrometry (MS)-based proteomics is the method of choice for the identification and quantification of thousands of proteins. However, the complete coverage of proteomes remains challenging due to the limited speed, sensitivity and resolution of current mass spectrometers.
The timsTOF Pro 2 uses the parallel accumulation serial fragmentation (PASEF®) acquisition method to provide extremely high speed and sensitivity, requiring only minimal sample amounts to reach new depths in proteomics.
被困的离子迁移谱法(蒂姆斯)是第一位and foremost a separation technique in the gas phase. This resolves sample complexity through an added dimension of separation in addition to high performance liquid chromatography (HPLC) and mass spectrometry, increasing peak capacity and confidence in compound characterization.
Equally important, the TIMS device also accumulates and concentrates ions of a given mass and mobility, enabling a unique increase in sensitivity and speed.
A near 100% duty cycle can be achieved with the dual-TIMS technology facilitating accumulation in the front section, while ions in the rear section are sequentially released depending on their mobility. This process of parallel accumulation serial fragmentation (PASEF®) enables collisional cross section (CCS) analysis.
CCS-enabled analysis opens up many further analytical possibilities, from greater certainty of compound identification to confident library matching and lower false discovery rates (FDRs) in large datasets.
Introducing the timsTOF Pro 2 mass spectrometer with our newest generation TIMS analyzer and a completely reworked stainless steel stacked ring ion guide (SRIG). The new design offers 3 times higher ion capacity. A new ion cooling multipole for ion pre-conditioning and improved ion injection into the quadrupole together with further simplified ion optics for more efficient ion transfer from the TIMS cell to the TOF analyzer, further maximize ion transfer and sensitivity both in MS and PASEF MS/MS.
The unique design of the TIMS cell means that ions are released from the second section of the TIMS analyzer depending on their mobility, while the further incoming ions can be accumulated in parallel in the first part of the TIMS analyzer.
This parallel accumulation technology achieves a duty cycle of nearly 100%, resulting in nearly no ion loss.
重新设计MS/MS技术以满足蛋白质组学的速度要求。使用被困的离子迁移率光谱法分离肽离子，洗脱（约100 ms），并在飞行的四极时间（QTOF）中检测到，生成TIMS MS热图。在平行的积累串行碎片中（Pasef®）方法使用了相同的TIM分离：四极杆在洗脱过程中分离出某种离子物种，并立即转移到下一个前体。父和片段光谱由迁移率值对齐。
PASEF®technology can achieve a sequencing speed of >100 Hz and the MS/MS spectra quality of the low abundant peptides can be increased by selecting them several times.
由Pasef提供动力的pro 2®提供> 100 Hz的测序速度而不会失去灵敏度或分辨率。这是通过将四极隔离质量窗口与来自TIMS漏斗的特定肽包装的洗脱时间同步的。
Minimal Cleaning Required
Many MS instruments used for proteomics applications require monthly cleaning when run 24 hours a day on large sample cohorts. Performance degradation is
noticeable even over shorter time periods. The superior robustness of the timsTOF Pro 2 means that the instrument can be run 24/7 over many weeks without noticeable loss of sensitivity or other performance metrics.
"Since we started to work with the TimsTOF Pro 26 months ago in February 2019, we have run more than 25000 LCMS samples, of which about 5000 have been non-depleted plasma digests. We had virtually zero downtime so far."
PaSER (Parallel Search Engine in Real-time) is a combined hardware and software solution enabling fully integrated real-time database searches and results based sample queue management.
Additionally, real-time LFQ quantification can also be performed across PaSER acquired data sets making the transition into quantitative proteomics instantaneous. Visualization of mobility offset mass aligned (MOMA) features using TIMS Viz allows the user to immediately identify and characterize isobaric and near-isobaric peptides that are only visible and identifiable by 4D-Omics.
dia-PASEF is both more sensitive and selective than traditional DIA approaches as it applies the PASEF principle to data independent acquisition, combining the advantages of DIA with the inherent ion efficiency of PASEF.
Making use of the correlation of molecular weight and CCS coded information from the dual-TIMS funnel, dia-PASEF enables most confident compound identification. Over the entire LC-MS/MS dia-PASEF runs a perfect data cuboid is created containing m/z, ion mobility (CCS), retention time and intensity.
使用可靠的SRIG（堆叠环离子指南）配置和新的优化标准DDA-PASEF方法Timstof Pro 2在单个Shot蛋白质组学中提供了前所未有的蛋白质组覆盖深度。从Aurora-25 cm柱中，从60分钟的梯度中的200 ng的Inhouse Hek胰蛋白酶摘要中，鉴定出7000多个蛋白质组和60,000肽。
TimstofPro 2 thus provides in-depth proteome coverages for everyday cell line proteome quantification experiments directly by database searching and matching between the runs without the need for any spectral library. Different database search strategies resulted in very comparable results. PaSER, enabling real time protein identifications, and MaxQuant resulted in similar ID numbers on both protein and peptide level.
In comparison with standard selected and parallel reaction monitoring (SRM and PRM), prm-PASEF increases the number of peptides that can be targeted in a single acquisition method, without compromising the selectivity or the sensitivity.
靶向质谱法（MS）是一种强大的技术，用于蛋白质组学实验中 - 例如，在大型样本队列中验证生物标志物候选物。与数据依赖性采集（DDA）和数据无关的获取（DIA）相比，这会提高灵敏度。该技术受到单个运行中测量的目标数量的必要折衷，液相色谱分离阶段的持续时间和整体灵敏度。只能通过在MS敏感性和选择性上进行更长的色谱分离或妥协来实现大量靶向肽的完整数据。
prm-PASEF increases the number of peptides that can be targeted in a single acquisition by benefiting from the 4th dimension of separation using Bruker’s timsTOF Pro 2 to improve selectivity and sensitivity, adding the speed of PASEF to increase the number of precursor targets.
Proteome quantification using low sample amounts is crucial for a growing number of biological applications such as specialized cells, rare cell populations, or fine needle aspiration tumor biopsies. Proteome quantification of such low sample amounts using a sensitive mass spectrometer is crucial. From 20 ng of HeLa (Pierce) peptides measured on Aurora-25 cm column in 30 minute gradients, PaSER – realtime search engine – identifies more than 4200 protein groups and close to 30,000 peptides.
Data independent acquisition using standard dia-PASEF methods provides reproducible identifications in multiple runs. Three different dia-PASEF window schemes allow for the quantification of close to 8000 protein groups and more than 70,000 peptide sequences in 60 minute gradients using Aurora-25cm columns, while demonstrating quantitative precision.
DIA-PASEF对Pro 2的高灵敏度，测序速度和可重复性甚至可以实现有限样品量的定量磷蛋白分析。从小鼠脑样品中获得的25μg总蛋白质可行，无标记的磷蛋白组定量可行。DIA-PASEF使用30个SPD（每天样品）EVOSEP方法对富集的磷酸肽进行分析，从而鉴定出三种富集重复的4473种独特的磷酸肽。这些结果对在针中的活检中的应用有了进一步的希望，并通过有关信号转导的信息来补充癌症蛋白质组学数据。结果由Stefan Tenzer教授提供。
Analyze cell signaling where sample amounts are limited
At the point of chromatographic co-elution, quantification of phospho-peptide (p-peptide) isomers is not possible in traditional proteomics approaches without CCS information due to the isobaric nature and signal overlay. PASEF analyses from standard 150 μg TiO2-based enrichment workflows identifies 27,768 phosphopeptides, as shown on the right and reveals the benefits of ion mobility separation with Mobility Offset Mass Aligned (MOMA). From 1946 identified co-eluting isomers, 20% could be fully separated by TIMS, enabling a better understanding of proximal protein phosphorylation sites.
"Besides its high sensitivity, a unique aspect of the timsTOF Pro instrument is its capability to resolve positional phosphorylation isomers in the gas phase, thus providing more detailed insight into signaling pathways."
For Research Use Only. Not for use in clinical diagnostic procedures.