timsTOF Pro 2

基于质谱（MS）的蛋白质组学是鉴定和定量数千种蛋白质的首选方法。但是，由于当前质谱仪的速度，灵敏度和分辨率有限，蛋白质组织的完整覆盖范围仍然具有挑战性。

The timsTOF Pro 2 uses the parallel accumulation serial fragmentation (PASEF^®）采集方法提供极高的速度和灵敏度，仅需要最小的样本量才能达到蛋白质组学的新深度。

捕获的离子迁移率（TIMS）是气相中首先是一种分离技术。除了高性能液相色谱（HPLC）和质谱法外，这可以通过分离的增加维度解决样品复杂性，从而增加了峰值容量和对复合表征的置信度。

Equally important, the TIMS device also accumulates and concentrates ions of a given mass and mobility, enabling a unique increase in sensitivity and speed.

通过双TIMS技术促进前部的积累，可以实现接近100％的占空比，而后部的离子则根据其迁移率依次释放。平行积累序列碎片的过程（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.

使用我们的最新一代TIMS分析仪和完全重新设计的不锈钢堆叠环离子指南（SRIG）引入TimStof Pro 2质谱仪。新设计可提供3倍的离子容量。一种用于离子预先调节和改进离子将四极的离子冷却多极以及进一步的简化离子光学元件，以从TIMS细胞到TOF分析仪的更有效的离子转移，进一步最大化离子传递和MS/ Pasef MS// Pasef MS/ Pasef MS/ Pasef多发性硬化症。

TIMS单元的独特设计意味着离子是根据TIMS分析仪的第二部分释放的，具体取决于其迁移率，而进一步的传入离子可以在TIMS分析仪的第一部分并行积累。
这种平行的积累技术实现了近100％的占空比，几乎没有离子损失。

Redesigned MS/MS technology to meet the speed requirements of proteomics. Peptide ions are separated using trapped ion mobility spectrometry, eluted (~ 100 ms) and detected in the quadrupole time of flight (QTOF), generating the TIMS MS heat map. In the parallel accumulation serial fragmentation (PASEF^®)方法使用相同的商旅分离:作为drupole isolates a certain ion species during its elution and immediately shifts to the next precursor. Parent and fragment spectra are aligned by mobility values.

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.

PASEF^®: the perfect fit for shotgun proteomics
The timsTOF Pro 2 powered by PASEF^®offers a sequencing speed of >100 Hz without losing sensitivity or resolution. This is achieved by synchronizing the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel.

需要最小的清洁
许多用于蛋白质组学应用的MS仪器需要每天24小时在大型样品队列上运行时每月清洁。性能退化是

在较短的时间段甚至更短的时间内。TimStof Pro 2的出色鲁棒性意味着可以在数周内24/7运行该仪器，而不会明显丧失灵敏度或其他性能指标。

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.

PaSER delivers results with uncompromising speed, including PTM searches. By utilizing GPU based searches, PaSER delivers the same results in real-time or offline mode without the need to utilize simplified algorithms or intermediates. This uncompromised search speed of PaSER allows you to have results on hand, seconds after the acquisition ends, Run & Done! PaSER effectively removes the data analysis bottleneck introduced by large sample cohorts and greater throughput.

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.

TIMS separation increases selectivity, excludes singly charged precursors from fragmentation and cleans up the sample by concentrating signals from noise.

DIA-PASEF利用分子量和CCS编码信息的CCS编码信息，可实现最自信的化合物识别。在整个LC-MS/MS DIA-PASEF中，创建一个完美的数据Cuboid，其中包含M/Z，离子迁移率（CCS），保留时间和强度。

With the robust SRIG (Stacked Ring Ion Guide) configuration and new optimized standard dda-PASEF methods timsTOF Pro 2 provides unprecedented depth of proteome coverage in single shot proteomics. From inhouse HEK tryptic digests of 200 ng in 60 minute gradients using an Aurora-25 cm columns, more than 7000 protein groups and 60,000 peptides were identified.

timsTOF Pro 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.

Targeted mass spectrometry (MS) is a powerful technique that is used in proteomics experiments – to verify biomarker candidates in large sample cohorts, for example. This increases sensitivity compared to data dependent acquisition (DDA) and data independent acquisition (DIA). This technique is limited by a necessary compromise between the number of targets measured in a single run, the duration of the liquid chromatography separation stage and the overall sensitivity. It is only possible to achieve complete data for a large number of targeted peptides either by running longer chromatography separation or compromising on MS sensitivity and selectivity.

PRM-PASEF通过使用Bruker的TimStof Pro 2从分离的第四维中受益，从而增加了可以在单个采集中靶向的肽数量，从而提高了选择性和敏感性，从而增加了PaseF的速度以增加前体目标的数量。

使用低样品量的蛋白质组定量对于越来越多的生物学应用，例如专业细胞，稀有细胞群或细针吸入肿瘤活检至关重要。使用敏感质谱仪对这种低样品量的蛋白质组定量至关重要。从30分钟的Aurora-25 cm柱上测量的20 ng HeLa（Pierce）肽，Paser - 实时搜索引擎 - 鉴定了4200多个蛋白质基团，接近30,000个肽。

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.

CCS-enabled quantification of proximal phosphorylation sites
The high sensitivity, sequencing speed and reproducibility of dia-PASEF on the timsTOF Pro 2 even enables quantitative phosphoproteomic analyses of limited sample amounts. Label-free quantification of phosphoproteomes is feasible from as little as 25 μg of total protein obtained from mouse brain samples. dia-PASEF analysis of enriched phosphopeptides using a 30 SPD (samples per day) Evosep method resulted in the identification of up to 4473 unique phosphopeptides across three enrichment replicates. These results hold further promise for the application in needle biopsies, complementing cancer proteogenomics data with information on signal transduction. Results are provided courtesy of Prof. Stefan Tenzer.

分析样品量有限的细胞信号传导
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.