Michael Steidel
@michaelsteidel.bsky.social
#Cellzome #TeamMassSpec #Proteomics opinions are my own
Pinned
Impact of Local Air Pressure on Ion Mobilities and Data Consistency in diaPASEF-Based High Throughput Proteomics
Data-independent acquisition (DIA) on ion mobility mass spectrometers enables deep proteome coverage and high data completeness in large-scale proteomics studies. For advanced acquisition schemes such as parallel accumulation serial fragmentation-based DIA (diaPASEF) stability of ion mobility (1/K0) over time is crucial for consistent data quality. We found that minor changes in environmental air pressure systematically affect the vacuum pressure in the TIMS analyzer, causing ion mobility shifts. By comparing experimental ion mobilities with historical weather data, we attributed observed drifts to fluctuations in the ground air pressure. Moderate air pressure changes of e.g. fifteen mbar induce ion mobility shifts of 0.025 Vs/cm2. These drifts negatively impact peptide quantification across consecutively acquired samples due to drift-dependent abundance changes and increased missing values for ions located at the boundaries of diaPASEF isolation windows, which cannot be corrected by postprocessing. To address this, we applied an in-batch mobility autocalibration feature on a run-wise basis, leading to full elimination of ion mobility drifts.
pubs.acs.org
Weather’s going wild, and now your mass spec data’s a mess too?
Coindidence? Nope!
We reveal how weather-driven air pressure fluctuations impact diaPASEF-based high troughput proteomics - and how to fix it!
Check out our new paper! #diaPASEF #Weatheromics
pubs.acs.org/doi/10.1021/...
Naive question: I thought targeted was about sensitivity -
why ng?
why ng?
January 3, 2026 at 11:33 AM
Naive question: I thought targeted was about sensitivity -
why ng?
why ng?
Reposted by Michael Steidel
It's called the Agilent 6495D.
January 3, 2026 at 10:32 AM
It's called the Agilent 6495D.
Not a conference, but worth flagging:
EMBO Practical Course “Targeted proteomics: advanced tools for biomedical research” Barcelona, 8–13 Nov 2026
Line-up not announced yet, but previous editions organized by @maccoss.bsky.social with invited speakers incl. Alexey Nesvizhskii and Vadim Demichev.
EMBO Practical Course “Targeted proteomics: advanced tools for biomedical research” Barcelona, 8–13 Nov 2026
Line-up not announced yet, but previous editions organized by @maccoss.bsky.social with invited speakers incl. Alexey Nesvizhskii and Vadim Demichev.
December 27, 2025 at 11:38 PM
Not a conference, but worth flagging:
EMBO Practical Course “Targeted proteomics: advanced tools for biomedical research” Barcelona, 8–13 Nov 2026
Line-up not announced yet, but previous editions organized by @maccoss.bsky.social with invited speakers incl. Alexey Nesvizhskii and Vadim Demichev.
EMBO Practical Course “Targeted proteomics: advanced tools for biomedical research” Barcelona, 8–13 Nov 2026
Line-up not announced yet, but previous editions organized by @maccoss.bsky.social with invited speakers incl. Alexey Nesvizhskii and Vadim Demichev.
Proteomic Ruler question:
In Wiśniewski et al., MCP 2014, the histone→DNA proxy seems implicit.
Is there any explicit reference stating that the Ruler uses only core histones (H2A/H2B/H3/H4) and excludes H1?
#proteomics #massspec
In Wiśniewski et al., MCP 2014, the histone→DNA proxy seems implicit.
Is there any explicit reference stating that the Ruler uses only core histones (H2A/H2B/H3/H4) and excludes H1?
#proteomics #massspec
December 14, 2025 at 1:41 PM
Proteomic Ruler question:
In Wiśniewski et al., MCP 2014, the histone→DNA proxy seems implicit.
Is there any explicit reference stating that the Ruler uses only core histones (H2A/H2B/H3/H4) and excludes H1?
#proteomics #massspec
In Wiśniewski et al., MCP 2014, the histone→DNA proxy seems implicit.
Is there any explicit reference stating that the Ruler uses only core histones (H2A/H2B/H3/H4) and excludes H1?
#proteomics #massspec
I agree MaxLFQ isn’t meant for absolute quantification.
But that still doesn’t explain the complete lack of correlation with UPS2.
From my experience, iBAQ and MaxLFQ usually correlate well (R² ~0.78, non related example dataset shown), suggesting they track the same MS1 signal.
But that still doesn’t explain the complete lack of correlation with UPS2.
From my experience, iBAQ and MaxLFQ usually correlate well (R² ~0.78, non related example dataset shown), suggesting they track the same MS1 signal.
December 7, 2025 at 11:00 PM
I agree MaxLFQ isn’t meant for absolute quantification.
But that still doesn’t explain the complete lack of correlation with UPS2.
From my experience, iBAQ and MaxLFQ usually correlate well (R² ~0.78, non related example dataset shown), suggesting they track the same MS1 signal.
But that still doesn’t explain the complete lack of correlation with UPS2.
From my experience, iBAQ and MaxLFQ usually correlate well (R² ~0.78, non related example dataset shown), suggesting they track the same MS1 signal.
As shown in the DIA-NN paper, the mobility term contributes only negligibly to the discriminant score, suggesting that measured CCS -even with good IM resolution - might simply be too affected by gas-phase ion–ion / ion–neutral interactions to provide a stable, high-specificity constraint …
December 6, 2025 at 2:16 PM
As shown in the DIA-NN paper, the mobility term contributes only negligibly to the discriminant score, suggesting that measured CCS -even with good IM resolution - might simply be too affected by gas-phase ion–ion / ion–neutral interactions to provide a stable, high-specificity constraint …
With a quadrupole we know exactly which precursor m/z window was isolated -the precursor mass is tightly defined with a well-characterised error. My question was whether CCS can provide anything close to that level of search-space restriction for database searching.
December 6, 2025 at 2:12 PM
With a quadrupole we know exactly which precursor m/z window was isolated -the precursor mass is tightly defined with a well-characterised error. My question was whether CCS can provide anything close to that level of search-space restriction for database searching.
My point rather was whether precursor CCS can actually constrain the search space during database searching not just what Da-equivalent tolerance it has.
December 6, 2025 at 2:04 PM
My point rather was whether precursor CCS can actually constrain the search space during database searching not just what Da-equivalent tolerance it has.
Asking for a friend: Is intrinsic specificity of CCS high enough to serve as an effective in silico precursor filter during database searching?
December 6, 2025 at 12:24 PM
Asking for a friend: Is intrinsic specificity of CCS high enough to serve as an effective in silico precursor filter during database searching?
“Very interesting! Do you know whether ProteomeSciences is already testing the new DXT tags with selected customers or collaborators, or is it still entirely in-house at this stage?”
November 20, 2025 at 11:54 AM
“Very interesting! Do you know whether ProteomeSciences is already testing the new DXT tags with selected customers or collaborators, or is it still entirely in-house at this stage?”
Reposted by Michael Steidel
Bonus, info about DIA multiplex tags, up to 30-plex:
"trademark DXT for our DIA multiplex tags...advances have been made in DXT multiplexing since ASMS with the number of tags increased from 6 to 11 and with the potential to
increase these to beyond 30"
"trademark DXT for our DIA multiplex tags...advances have been made in DXT multiplexing since ASMS with the number of tags increased from 6 to 11 and with the potential to
increase these to beyond 30"
November 18, 2025 at 10:09 AM
Bonus, info about DIA multiplex tags, up to 30-plex:
"trademark DXT for our DIA multiplex tags...advances have been made in DXT multiplexing since ASMS with the number of tags increased from 6 to 11 and with the potential to
increase these to beyond 30"
"trademark DXT for our DIA multiplex tags...advances have been made in DXT multiplexing since ASMS with the number of tags increased from 6 to 11 and with the potential to
increase these to beyond 30"
Reposted by Michael Steidel
Fantastic project led by @bo-wen.bsky.social. Excited to see the future uses of AI and transfer learning in proteomics. #massspec #proteomics
www.nature.com/articles/s41...
www.nature.com/articles/s41...
Carafe enables high quality in silico spectral library generation for data-independent acquisition proteomics - Nature Communications
Accurate spectral libraries are essential for analyzing data-independent acquisition (DIA) proteomics data. Here, the authors present Carafe, which trains on DIA data to build experiment-specific spec...
www.nature.com
November 6, 2025 at 3:49 PM
Fantastic project led by @bo-wen.bsky.social. Excited to see the future uses of AI and transfer learning in proteomics. #massspec #proteomics
www.nature.com/articles/s41...
www.nature.com/articles/s41...
Which LC & Flow?
October 7, 2025 at 4:38 PM
Which LC & Flow?
Surprised that u go so low. With EvoSep 24 min method we can load lots more on our Ultra2 until reaching saturation especially with ICC2.0
October 7, 2025 at 2:56 PM
Surprised that u go so low. With EvoSep 24 min method we can load lots more on our Ultra2 until reaching saturation especially with ICC2.0
How much are you loading per injection? Is ICC 2.0 enabled on the Ultra2? And which library are you using?
October 7, 2025 at 6:01 AM
How much are you loading per injection? Is ICC 2.0 enabled on the Ultra2? And which library are you using?
True — but the odd part is that the Human Reference Proteome is not really ‘canonical only’. Non-canonical entries from TrEMBL are included, yet the curated SwissProt isoforms are missing default. That’s what undermines the idea of a high-quality reference set.
October 2, 2025 at 6:34 AM
True — but the odd part is that the Human Reference Proteome is not really ‘canonical only’. Non-canonical entries from TrEMBL are included, yet the curated SwissProt isoforms are missing default. That’s what undermines the idea of a high-quality reference set.
On a separate note: I was surprised to find that none of the non-canonical SwissProt isoforms are included in the official human reference proteome (UP000005640).
Anyone know what’s going on here? 🤔
#proteomics #bioinformatics @pwilmarth.bsky.social il
Anyone know what’s going on here? 🤔
#proteomics #bioinformatics @pwilmarth.bsky.social il
October 1, 2025 at 3:13 PM
On a separate note: I was surprised to find that none of the non-canonical SwissProt isoforms are included in the official human reference proteome (UP000005640).
Anyone know what’s going on here? 🤔
#proteomics #bioinformatics @pwilmarth.bsky.social il
Anyone know what’s going on here? 🤔
#proteomics #bioinformatics @pwilmarth.bsky.social il
By the way.. 43% of current TREMBL entries will be dropped soon anyway insideuniprot.blogspot.com/2025/06/capt...
Capturing the Diversity of Life - Reorganizing the Protein Space in UniProtKB
Advances in genome sequencing technology means that large-scale efforts such as the Earth Biogenome project and the Darwin Tree of Life ...
insideuniprot.blogspot.com
September 30, 2025 at 2:22 PM
By the way.. 43% of current TREMBL entries will be dropped soon anyway insideuniprot.blogspot.com/2025/06/capt...
Hey #TeamMassSpec,
Many non-human proteomics studies still search against taxon-filtered FASTAs.
❌ Redundant sequences
❌ Inflated search space
✅ Reference proteomes cut redundancy, improve annotation, and make results comparable.
👉 Time to move beyond taxon filters. #proteomics #massspec #uniprot
Many non-human proteomics studies still search against taxon-filtered FASTAs.
❌ Redundant sequences
❌ Inflated search space
✅ Reference proteomes cut redundancy, improve annotation, and make results comparable.
👉 Time to move beyond taxon filters. #proteomics #massspec #uniprot
September 30, 2025 at 2:21 PM
Hey #TeamMassSpec,
Many non-human proteomics studies still search against taxon-filtered FASTAs.
❌ Redundant sequences
❌ Inflated search space
✅ Reference proteomes cut redundancy, improve annotation, and make results comparable.
👉 Time to move beyond taxon filters. #proteomics #massspec #uniprot
Many non-human proteomics studies still search against taxon-filtered FASTAs.
❌ Redundant sequences
❌ Inflated search space
✅ Reference proteomes cut redundancy, improve annotation, and make results comparable.
👉 Time to move beyond taxon filters. #proteomics #massspec #uniprot
Reposted by Michael Steidel
Without #2, a lower ion count is needed just to be sure that the full MS range is scanned, but with more accurate ion counts, you can go to the max S/N without losing ions on the edges.
This could also work for the Orbitrap Astral.
Bonus: DIAPASEF on Thermo - patentscope.wipo.int/search/en/de...
This could also work for the Orbitrap Astral.
Bonus: DIAPASEF on Thermo - patentscope.wipo.int/search/en/de...
September 28, 2025 at 1:25 PM
Without #2, a lower ion count is needed just to be sure that the full MS range is scanned, but with more accurate ion counts, you can go to the max S/N without losing ions on the edges.
This could also work for the Orbitrap Astral.
Bonus: DIAPASEF on Thermo - patentscope.wipo.int/search/en/de...
This could also work for the Orbitrap Astral.
Bonus: DIAPASEF on Thermo - patentscope.wipo.int/search/en/de...
Reposted by Michael Steidel
With 𝗗𝗜𝗔-𝗡𝗡 𝟮.𝟯.𝟬 Preview (Academia-only for now), we showcase the transformative new capabilities that have been developed in the past months. Download: github.com/vdemichev/Di...
September 26, 2025 at 9:52 AM
With 𝗗𝗜𝗔-𝗡𝗡 𝟮.𝟯.𝟬 Preview (Academia-only for now), we showcase the transformative new capabilities that have been developed in the past months. Download: github.com/vdemichev/Di...
Thanks @pwilmarth.bsky.social - also included the less-redundant "one protein per gene" db here ...Has anybody assessed potential benefits of the reduced search space on sensitivity?
September 25, 2025 at 9:21 PM
Thanks @pwilmarth.bsky.social - also included the less-redundant "one protein per gene" db here ...Has anybody assessed potential benefits of the reduced search space on sensitivity?
aaah guess its "hidden" there :)
September 25, 2025 at 1:39 PM
aaah guess its "hidden" there :)
Great thanks! Where can I find the one protein per gene option?
September 25, 2025 at 1:34 PM
Great thanks! Where can I find the one protein per gene option?
Hey #TeamMassSpec,
When you run proteomics on non-human species (mouse, rat, macaque, etc.) — which protein FASTA do you prefer?
Taxonomy-filtered UniProt (all entries)
Reference proteome (SwissProt+TrEMBL)
Ensembl/GENCODE
Something else?
When you run proteomics on non-human species (mouse, rat, macaque, etc.) — which protein FASTA do you prefer?
Taxonomy-filtered UniProt (all entries)
Reference proteome (SwissProt+TrEMBL)
Ensembl/GENCODE
Something else?
September 25, 2025 at 12:41 PM
Hey #TeamMassSpec,
When you run proteomics on non-human species (mouse, rat, macaque, etc.) — which protein FASTA do you prefer?
Taxonomy-filtered UniProt (all entries)
Reference proteome (SwissProt+TrEMBL)
Ensembl/GENCODE
Something else?
When you run proteomics on non-human species (mouse, rat, macaque, etc.) — which protein FASTA do you prefer?
Taxonomy-filtered UniProt (all entries)
Reference proteome (SwissProt+TrEMBL)
Ensembl/GENCODE
Something else?