Expanding the horizons of single cell research

timstof scp

用于定量单细胞生物学研究的TIMSTOF SCP与无偏的深单细胞4D-蛋白质组学™,免疫肽学,表现蛋白质组学和PTM分析相辅相成,以补充SCRNA-SEQ。扩展单细胞研究的视野。

重新定义单细胞蛋白质组学

Discover true proteome heterogeneity

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Destaques

timstof scp

Expanding the horizons of single cell research

超高灵敏度
新型离子源几何形状的突破性设计,其离子转移高达五倍和超高鲁棒性。
数据完整性
Data independent acquisition–parallel accumulation serial fragmentation (dia-PASEF) pushes the limits of reproducible quantification to pave the way to study cell heterogeneity at scale.
Acquisition Speed
高采集速度与DIA-PASEF敏感性相结合,可以在短液相色谱(LC)下进行分析,而无需色谱稀释,样品量非常低。
Robustness
双向异构反射到被困的离子迁移率光谱法(TIMS)可以实现数千个样品的常规操作,而无需清洁。

características

timstof scp

重新定义单细胞蛋白质组学

高级离子光学和PASEF研究单个细胞的细胞异质性和生物学
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。

Radically improved ion transfer

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.

较大的毛细血管产生超高的灵敏度,并且额外的正交离子反射和随后的漏斗提供了一个单独的差异泵送阶段,从而保持了TimStof仪器系列预期的系统稳健性。

Dramatic improvements in proteomics performance

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。

双TIMS,启用CCS的分析

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.

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。

Ideal for immunopeptidomics and other enrichment workflows

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.

Pasef®

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®)技术实现> 100 Hz的测序速度。使用Pasef®increases the MS/MS spectra quality of the low abundant peptides by selecting them several times.

Pasef®:非常适合shot弹枪蛋白质组学
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.

Benefícios

PaSER Run & Done – real time quality control for unbiased single cell analysis

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.

MaxQuant/Perseus and PEAKS Studio data processing

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

布鲁克’s MaxQuant software has been adapted to manage 4-dimensional (4D) features in the space spanned by retention time, ion mobility, mass, and signal

强度。这使肽,蛋白质和翻译后修饰的鉴定和定量有益。

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。

aplicações

Ultra-high sensitivity 4D-Proteomics with dia-PASEF

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。

探索肿瘤微环境

了解TME及其浸润的免疫细胞可以被认为是影响疾病进展,治疗反应和患者生存的关键步骤。由于其高灵敏度,SCP的TIMSTOF可以使FFPE组织样品中的激光捕获显微解剖(LMD)对细胞进行足够的蛋白质组深度。右图显示了典型的工作流程。

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。
结果由Matthias Mann教授提供(doi::https://doi.org/10.1101/2020.12.22.423933

网络研讨会

Testemunhos

"I always said that single cell proteomics would not happen in my lifetime, but I'm happy to have been proven wrong"

Prof. Matthias Mann, Ph.D., Director, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Martinsried, Germany

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