pro 2

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.

pro 2的timstof Pro 2使用平行的累积序列碎片（Pasef^®) acquisition method to provide extremely high speed and sensitivity, requiring only minimal sample amounts to reach new depths in proteomics.

Trapped ion mobility spectrometry (TIMS) is first 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.

同样重要的是，TIMS设备还积累了给定质量和迁移率的浓缩离子，从而实现了灵敏度和速度的独特提高。

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^®）启用碰撞横截面（CCS）分析。

启用CCS的分析从更大的复合识别确定性到自信的图书馆匹配和较低的错误发现率（FDR）开辟了许多进一步的分析可能性。

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.

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^®技术可以实现> 100 Hz的测序速度，并且可以通过选择几次来提高低丰富肽的MS/MS光谱质量。

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.

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.

PASER（实时并行搜索引擎）是一种组合的硬件和软件解决方案，可实现完全集成的实时数据库搜索和基于结果的样本队列管理。

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.

此外，还可以在Paser获得的数据集中进行实时LFQ定量，从而使该过渡为定量蛋白质组学瞬时。使用TIMS的迁移率偏移质量对齐（MOMA）特征的可视化使用户可以立即识别和表征仅由4D-omics可见和可识别的同位和近异构肽。

DIA-PASEF比传统DIA方法更敏感和选择性，因为它将PaseF原理应用于数据无关的获取，将DIA的优势与Pasef的固有离子效率相结合。

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

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.

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.

因此，timstof pro 2通过数据库搜索和匹配在运行之间，无需任何光谱库提供了直接通过数据库搜索和匹配的日常细胞系蛋白质量化实验的深入蛋白质组覆盖范围。不同的数据库搜索策略导致了非常可比的结果。Paser，可以实现实时蛋白质鉴定和最大的蛋白质鉴定，从而在蛋白质和肽水平上都产生了相似的ID数。

与标准选择的和平行的反应监测（SRM和PRM）相比，PRM-PASEF增加了可以以单个采集方法靶向的肽的数量，而不会损害选择性或敏感性。

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 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.

使用标准DIA-PASEF方法的数据独立采集可在多个运行中提供可重复的识别。三种不同的DIA-PASEF窗口方案允许使用Aurora-25cm列在60分钟的梯度中定量接近8000个蛋白质组和70,000多个肽序列，同时证明了定量精度。

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.

Analyze cell signaling where sample amounts are limited
在色谱共同排列时，在没有CCS信息的传统蛋白质组学方法中，由于等质性质和信号叠加层而无法进行磷酸肽（P肽）异构体的定量。如右图所示，来自标准150μgTiO2的富集工作流程的PASEF分析可识别27,768个磷酸肽，并揭示了离子迁移率分离及其迁移率偏移质量（MOMA）的好处。从1946年确定的共同洗脱异构体，可以通过TIMS完全分离20％，从而可以更好地了解近端蛋白质磷酸化位点。