{"id":15596,"date":"2023-08-14T15:07:56","date_gmt":"2023-08-14T15:07:56","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/?p=15596"},"modified":"2023-10-13T21:03:39","modified_gmt":"2023-10-13T21:03:39","slug":"single-cell-sensitivity-and-low-load-proteomics-q-and-a-for-the-novice","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/single-cell-sensitivity-and-low-load-proteomics-q-and-a-for-the-novice\/","title":{"rendered":"Single-Cell Sensitivity and Low-Load Proteomics: Q&A for the Novice"},"content":{"rendered":"\n

Single-cell proteomics (SCP) is a growing field that has prompted numerous technological advances in cell isolation, sample preparation, liquid chromatography (LC), mass spectrometry (MS) and data analysis for low sample amounts.<\/p>\n\n\n\n

These advancements occur because, while bulk sample studies provide detailed information about the system\u2019s average state, they overlook the emergent properties derived from biological heterogeneity, which can be observed only by looking into the state of individual cells.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Bulk-omics studies obscure variability between cells and provide a misleading sense of uniformity in their study systems. In highly heterogeneous systems \u2014 such as brains with multiple specialized cell types \u2014 these studies can produce average readings that do not represent any single cell found within the system, misdirecting research conclusions.<\/p>\n\n\n\n

Additionally, in areas like clinical and translational proteomics where sample amounts are limited, getting more out of limited samples, through technological advancements, is a requirement.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

How has low load\/SCP become more mainstream?<\/h3>\n\n\n\n

The introduction of commercially available low-load columns allowed for better separation of picograms of proteins, which are typically extracted from single cells. The \u00b5PAC Neo HPLC columns introduced the low-load version and have been a game changer for creating sharp peaks with minimal sample loss. The ordered capillary structure of these columns leads to excellent chromatography \u2014 a boon for all samples \u2014 but especially low peptide loads with the reproducibility to analyze thousands of single cells\u2019 sharper peaks with low loss. This allows hyper concentration of low abundant peptide on the column for better downstream detection by the mass spectrometer.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

The development of FAIMS (high field asymmetric ion mobility spectrometry) has also played a significant role in SCP. The FAIMS Pro Duo interface can eliminate high abundant background ions, allowing for detection of low abundant peptide signals, which tends to occur when dealing with low sample amounts. The FAIMS Pro Duo is also completely external to the mass spectrometer and can be removed, attached, or cleaned easily by the user.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

The base peak chromatograms above show the difference overall when injecting just 50 pg of HeLa standard digest onto the Orbitrap Ascend Tribrid MS.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

The data shown above highlights how the signal-to-noise (S\/N) ratio is affected by FAIMS gas-phase separation for individual peptides from the same 50 pg HeLa injection. The selected peptide experienced a 10-fold increase in S\/N and the fragmentation spectrum is much improved as well.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Is there any change to the Thermo Scientific Orbitrap MS system needed to run SCP?<\/strong><\/h3>\n\n\n\n

No, there is no configuration change needed to the MS. However, the system can be optimized for SCP in the methods. All current generation platforms have built-in template methods, which recommend parameters to follow and to adjust from as a great starting point. Highly sensitive mass spectrometers, such as the Orbitrap Ascend Tribrid MS, can even perform some low-load and single-cell experiments without the need for FAIMS. Still, higher identifications and better data quality are achieved when any system is coupled with FAIMS.<\/p>\n\n\n\n

What changes compared to a regular (i.e., 200 ng injection amount) MS method are most vital?<\/strong><\/h3>\n\n\n\n

Low injection amounts of sample tend to require longer ion injection time to ensure there will be enough precursor collected and fragmented to give meaningful data. This parameter is usually one of the most important settings in an Orbitrap mass spectrometer but plays a much more significant role in SCP. The ability to accurately accumulate ions to increase sensitivity with Automatic Gain Control (AGC) is a key benefit of Orbitrap and linear ion trap instruments, which other solutions cannot achieve.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

We also recommend being mindful of the FAIMS compensation voltage (CV). SCP experiments require very short LC-MS running time; therefore, we recommend using fewer compensation voltages per experiment. Generally, we recommend one compensation voltage for gradients 30 minutes or less. The voltage can be optimized, but generally we recommend a CV of -50 V.<\/p>\n\n\n\n

Which MS method is easiest to implement for SCP\/low load?<\/strong><\/h3>\n\n\n\n

The single-cell proteomics community has adopted data-independent acquisition (DIA) based label-free relative quantitative approaches. This approach is ideal for dealing with limited sample amounts and the short MS run used in SCP.  Another benefit of DIA is that there are theoretically no missing values between runs because all precursors should be fragmented and identified. This is different from data-dependent acquisition (DDA) approaches.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

What is DirectDIA and how does it help?<\/strong><\/h3>\n\n\n\n

DIA data has traditionally been analyzed using spectral libraries built from DDA experiments. However, the experimental design must be carefully considered since a library must be available or created for your specific sample. This was a limiting factor in the adoption of DIA for LC-MS analysis.<\/p>\n\n\n\n

DirectDIA is a data analysis scheme where the DIA experiment itself produces a peptide library from a prediction based on a FASTA file provided to the software. There are benefits and downsides to using this approach. The scientist interested in SCP needs to know how different the results can be when using different processing software. By using DirectDIA, though, there is no need to spend long periods of time collecting and analyzing DDA-based data to build a library to search the DIA data \u2014 saving resources and time.<\/p>\n\n\n\n

What kind of results should be expected?<\/strong><\/h3>\n\n\n\n

Using Orbitrap MS technology for SCP or a limited sample amount is simple and seamless. There are little to no changes needed from a general proteomics setup (unless adding FAIMS, an external hardware with plug-and-play connectivity right onto the inlet of the instrument). Our sensitive and versatile instruments produce world class results for any experiment type, from intact complexes to accurate quantitation.<\/p>\n\n\n\n

The Orbitrap Ascend Tribrid MS is our most versatile system. It is designed for any type of experiment, not only SCP and low load types, and yet it can outperform other instruments designed explicitly for SCP work.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Figure shows the results of the workflow outlined above using \u00b5PAC Neo HPLC column<\/a>, FAIMS Pro Duo interface, and a DirectDIA analysis using a 40 samples per day method, injecting 250 pg of standard HeLa digest on both the Thermo Scientific\u2122 Orbitrap Exploris\u2122 480 MS<\/a> and the Orbitrap Ascend Tribrid MS<\/a> compared to published data with as similar an analysis as possible from a competitor\u2019s platform.<\/p>\n\n\n\n

Learn more about Thermo Fisher’s single-cell solutions.<\/a> <\/p>\n","protected":false},"excerpt":{"rendered":"

Single-cell proteomics (SCP) is a growing field that has prompted numerous technological advances in cell isolation, sample preparation, liquid chromatography (LC), mass spectrometry (MS) and data analysis for low sample amounts. These advancements occur because, while bulk sample studies provide detailed information about the system\u2019s average state, they overlook the emergent properties derived from biological<\/p>\n","protected":false},"author":1327,"featured_media":15611,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_monsterinsights_skip_tracking":false,"_genesis_hide_title":false,"_genesis_hide_breadcrumbs":false,"_genesis_hide_singular_image":false,"_genesis_hide_footer_widgets":false,"_genesis_custom_body_class":"","_genesis_custom_post_class":"","_genesis_layout":"","_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":""},"categories":[82],"tags":[85,24,1765],"division":[],"class_list":{"0":"post-15596","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-proteomics","8":"tag-lc-ms","9":"tag-orbitrap","10":"tag-single-cell-proteomics","11":"entry"},"_selected_authors":"","_selected_reviewers":"","acf":[],"yoast_head":"\nSingle-Cell 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