timstof scp

Advanced ion optics and PASEF to investigate cell heterogeneity and biology from a single cell
Mass spectrometric proteomics has become a staple of modern research in understanding biological function and disease mechanisms. Healthy or diseased tissues that seem homogenous are composed of cells with a variety of different proteomes. The challenge of deciphering the proteomes in each single cell – the cell heterogeneity – holds the key to fully understanding its function.
SCP的TIMSTOF提供了一个从根本上改进的离子源概念。与平行的积累串行碎片结合（Pasef^®）采集方法，它提供了极高的速度和灵敏度，可以解决单个细胞的蛋白质组织或在形态学或功能上相似的一些细胞中的转换修饰。

The timsTOF SCP features a modified ion source geometry that includes 1 mm capillary identification for five times higher ion transfer into an additional higher pressure stage ion funnel and 8-stage differentially pumped vacuum system.

The larger capillary yields ultra-high sensitivity and the additional orthogonal ion reflection and subsequent funnel offers a separate, differentially pumped stage, maintaining the system robustness expected from the timsTOF instrument series.

SCP TIMSTOF提供了离子传输到源的改进，同时还可以通过增加更高的压力真空阶段来保持稳健性。这导致离子电流几乎改善。当与Evosep One Whisper方法结合使用时，以100 nl/min的流量运行

Pasef方法，与先前的高流量结果相比，通过EVOSEP ONE的敏感性增长约为100倍。这可以在真正的单细胞水平上实现无偏的蛋白质组学，其可重复性，鲁棒性和每个细胞的覆盖率良好，覆盖率约为1500个蛋白质。

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。

除了无偏的真实单细胞蛋白质组学应用外，SCP的TIMSTOF还对涉及蛋白质组富集肽的工作流程提供了出色的敏感性。免疫肽组学研究从血浆或组织的免疫肽纯化开始。

Since immunopeptides are present at relatively low abundance in these samples, the timsTOF SCP is ideal for immunpeptidomics for neo-antigen discovery where the available material is limited, as in needle biopsies. The timsTOF SCP also has the sensitivity to revolutionize the use of phosphoproteomics for the study of signaling pathways in cancer.

使用被困的离子迁移率光谱法（TIMS）分离肽离子，洗脱（〜100 ms），并在飞行的四倍时间（QTOF）中检测到，生成TIMS MS热图。在Pasef中^®方法在洗脱过程中，将某个离子物种隔离并立即转移到下一个前体。父和片段光谱由迁移率值对齐。

平行积累的序列碎片（Pasef^®）technology achieves a sequencing speed of >100 Hz. Using PASEF^®increases the MS/MS spectra quality of the low abundant peptides by selecting them several times.

Pasef^®: the perfect fit for shotgun proteomics
The timsTOF SCP 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 as well as the collision energy in the collision cell.

The timsTOF SCP allows the analysis of hundreds of samples with minimal sample load (<200 ng) at sequencing speeds exceeding 100 Hz and with uncompromised proteomic depth. This has changed the way proteomics is studied, but the increased data requires a new level of data analysis.

Data analysis is a common bottleneck in many workflows. Modern analytical methods result in hundreds of lines of data generated by the timsTOF SCP. Bruker has introduced real-time database search capabilities - parallel database search engine in real-time (PaSER) which removes this hurdle. Using PaSER, as soon as a LC-MS run is completed, results are available – effectively Run & Done.

An open-file data format allows researchers to work directly with raw data and use industry leading software of their choice.

Maxquant软件已适应用于管理空间中的4维（4D）特征，该功能由保留时间，离子迁移率，质量和信号强度跨越。从PASEF中，这有益于肽，蛋白质和翻译后修饰的识别和定量^®和diam-pasef^®data.

Peaks Studio将从头测序与传统数据库搜索结合在一起，并经过优化用于处理TimStof原始数据。

高级蛋白质学处理软件解决方案的数量读取了原始数据，包括Biognosys的Spectronaut和Spectromine软件以及Skyline，GenEDATA，GenEDATA，蛋白质指标，Matrix Science，DIA-NN和MSFragger的吉祥物，蛋白质指标，吉祥物酿酒师的软件。

基于CCS的分析，用于自信识别
TimScontrol允许自定义Dia-Pasef窗口方案专注于

感兴趣的离子。调整质量隔离宽度，TIMS范围和周期时间，可以使DIA-PASEF适应不同的色谱法。

将低流量的液体色谱与埃弗斯普（Evosep One）系统的低语与dia-pasef在timstof scp上的高灵敏度结合在一起，从500 pg的细胞摘要中鉴定出超过2000蛋白质，从250 pg中鉴定出1500 pg的蛋白质，证明了所需的敏感性，证明了所需的灵敏度真正的单细胞蛋白质组学。从250 pg鉴定出的蛋白质覆盖了大约4个数量级的丰度范围，从而在单细胞水平上实现了定量蛋白质组分析。

了解其infiltrati开心的在一起ng immune cells can be considered a crucial step to influence disease progression, treatment response and patient survival. Due to its high sensitivity the timsTOF SCP enables sufficient proteome depth on cells excised by laser capture microdissection (LMD) from FFPE tissue samples. A typical workflow is shown in the figure on the right.

Cellular markers were used to identify melanoma cancer cells in the tumor mass and those closely related to the stroma for subsequent isolation of both populations by LMD and unbiased 4D-Proteomics analysis on a timsTOF SCP instrument.

关键发现：富集分析表明，中央和周围黑色素瘤细胞之间的差异调节蛋白质具有疾病亚型的潜力以指导临床决策。
Results are provided by courtesy of Prof. Matthias Mann (doi:https://doi.org/10.1101/2020.12.22.423933）