timsTOF SCP

Advanced ion optics and PASEF to investigate cell heterogeneity and biology from a single cell
质谱蛋白质组学已成为理解生物学功能和疾病机制的现代研究的主要研究。看起来同质的健康或患病的组织由具有多种不同蛋白质组的细胞组成。在每个单个细胞中解密蛋白质组的挑战（细胞异质性）是完全理解其功能的关键。
timsTOF SCP提供x射线检验cally improved ion source concept. Combined with parallel accumulation serial fragmentation (PASEF^®) acquisition methods, it provides extremely high speed and sensitivity to tackle proteomes of single cells or post translational modifications in a few cells that are morphologically or functionally similar.

TIMSTOF SCP具有修饰的离子源几何形状，其中包括1毫米毛细管识别，其离子转移到额外的高压期离子漏斗和8阶段差分抽水的真空系统中。

更大的毛细管收益率超高灵敏度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.

The timsTOF SCP offers improvements in ion transmission into the source while maintaining robustness with an added higher pressure vacuum stage. This results in an almost 5x improvement in ion current. When combined with the Evosep One Whisper method running at 100 nL/min flow rate, and the dia-

PASEF method, sensitivity gains of about 100X over previous high-flow results with the Evosep One are achieved. This enables unbiased proteomics at the true single cell level with good reproducibility, robustness and coverage of about 1500 proteins per cell for the first time.

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.

Besides unbiased true single cell proteomics applications, the timsTOF SCP also offers outstanding sensitivity for workflows that involve enrichment of peptides from the proteome. Immunopeptidomics studies start with purification of immunopeptides from plasma or tissue.

由于这些样品中的免疫肽存在相对较低的丰度，因此SCP非常适合用于新抗原发现的免疫肽疗法，而可用材料受到限制的新抗原，如针刺活检。SCP的TIMSTOF还具有革命性使用磷蛋白组学用于研究癌症信号通路的敏感性。

Peptide ions are separated using trapped ion mobility spectrometry (TIMS), eluted (~ 100 ms) and detected in the quadropole time of flight (QTOF), generating the TIMS MS heat map. In the PASEF^®method the same TIMS separation is used with the quadrupole isolating a certain ion species during its elution and immediately shifting to the next precursor. Parent and fragment spectra are aligned by mobility values.

The parallel accumulation serial fragmentation (PASEF^®) technology achieves a sequencing speed of >100 Hz. Using PASEF^®通过选择多次，可提高低丰富肽的MS/MS光谱质量。

PASEF^®: the perfect fit for shotgun proteomics
由PASEF提供动力的SCP的TIMSTOF^®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以及碰撞电池中的碰撞能量同步的。

SCP的TIMSTOF允许在超过100 Hz且毫不妥协的蛋白质组学深度的测序速度下分析数百种样品载荷（<200 ng）的样品。这改变了研究蛋白质组学的研究方式，但是增加的数据需要新的数据分析水平。

数据分析是许多工作流程中常见的瓶颈。现代分析方法导致由SCP产生的数百条数据线。布鲁克（Bruker）已引入实时数据库搜索功能 - 实时（PASER）并行数据库搜索引擎，该引擎删除了此障碍。使用PASER，一旦完成LC-MS运行，就可以有效地运行并完成结果。

开放文件格式使研究人员可以直接与原始数据合作，并使用其选择的行业领先软件。

Bruker的最大软件已改编成管理在保留时间，离子移动性，质量和信号的空间中的4维（4D）功能

intensity. This benefits the identification and quantification of peptides, proteins, and posttranslational modifications.

PEAKS Studio combines de novo sequencing with traditional database searches and is optimized for processing timsTOF raw data.

CCS-enabled analysis for confident identifications
timsControl allows customization of the dia-PASEF window scheme to focus on

the ions of interest. Adjusting the mass isolation width, TIMS range and cycle time allows adaptation of dia-PASEF to different chromatography methods.

Combining low flow liquid chromatography on the Evosep One system with Whisper with the high sensitivity of dia-PASEF on the timsTOF SCP, over 2000 proteins were identified from 500 pg of cell digest and 1500 proteins were identified from 250 pg, demonstrating the sensitivity needed for true single cell proteomics. The proteins identified from 250 pg covered an abundance range of about 4 orders of magnitude, enabling quantitative proteome analysis at single cell level.

Understanding the TME together with its infiltrating 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.

细胞标记用于鉴定肿瘤质量中的黑色素瘤癌细胞，以及与基质密切相关的癌细胞，以随后通过LMD和无偏见的SCP仪器分离群体和无偏见的4D蛋白质组学分析。

Key Finding:Enrichment analysis revealed differentially regulated proteins between central and peripheral melanoma cells with potential for disease subtyping to guide clinical decision-making.
Results are provided by courtesy of Prof. Matthias Mann (doi:https://doi.org/10.1101/2020.12.22.423933)