MALDI MSI Protocol for Spatial Bottom-Up Proteomics at Single-Cell Resolution

Overview

Journal J Proteome Res

Publisher American Chemical Society

Specialty Biochemistry

Date 2024 Oct 24

PMID 39447324

Authors

Andrej Grgic

Eva Cuypers

Ludwig J Dubois

Shane R Ellis

Ron M A Heeren

Affiliations

Soon will be listed here.

Abstract

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) started with spatial mapping of peptides and proteins. Since then, numerous bottom-up protocols have been developed. However, achievable spatial resolution and sample preparation with many wet steps hindered the development of single cell-level workflows for bottom-up spatial proteomics. This study presents a protocol optimized for MALDI-MSI measurements of single cells within the context of their 2D culture. Sublimation of CHCA, followed by a dip in ice-cold ammonium phosphate monobasic (AmP), produced peptide-rich mass spectra while maintaining matrix crystal sizes around 400 nm. This enables MALDI-MSI imaging of proteins in single cells grown on an ITO slide with a throughput of approximately 7800 cells per day. 89 peptide-like features corresponding to a single MDA-MB-231 breast cancer cell were detected. Furthermore, by combining the MALDI-MSI data with LC-MS/MS data obtained on cell pellets, we have successfully identified 24 peptides corresponding to 17 proteins, including actin, vimentin, and transgelin-2.

Citing Articles

Molecular Profiling of Glioblastoma Patient-Derived Single Cells Using Combined MALDI-MSI and MALDI-IHC.

Krestensen K, Hendriks T, Grgic A, Derweduwe M, De Smet F, Heeren R Anal Chem. 2025; 97(7):3846-3854.

PMID: 39932302 PMC: 11866282. DOI: 10.1021/acs.analchem.4c03821.

References

Cuypers E, Claes B, Biemans R, Lieuwes N, Glunde K, Dubois L . 'On the Spot' Digital Pathology of Breast Cancer Based on Single-Cell Mass Spectrometry Imaging. Anal Chem. 2022; 94(16):6180-6190. PMC: 9047448. DOI: 10.1021/acs.analchem.1c05238. View

Velickovic D, Sharma K, Alexandrov T, Hodgin J, Anderton C . Controlled Humidity Levels for Fine Spatial Detail Information in Enzyme-Assisted -Glycan MALDI MSI. J Am Soc Mass Spectrom. 2022; 33(8):1577-1580. DOI: 10.1021/jasms.2c00120. View

Stauber J, MacAleese L, Franck J, Claude E, Snel M, Kaletas B . On-tissue protein identification and imaging by MALDI-ion mobility mass spectrometry. J Am Soc Mass Spectrom. 2009; 21(3):338-47. DOI: 10.1016/j.jasms.2009.09.016. View

Niehaus M, Soltwisch J, Belov M, Dreisewerd K . Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution. Nat Methods. 2019; 16(9):925-931. DOI: 10.1038/s41592-019-0536-2. View

Spraggins J, Djambazova K, Rivera E, Migas L, Neumann E, Fuetterer A . High-Performance Molecular Imaging with MALDI Trapped Ion-Mobility Time-of-Flight (timsTOF) Mass Spectrometry. Anal Chem. 2019; 91(22):14552-14560. PMC: 7382025. DOI: 10.1021/acs.analchem.9b03612. View

Scupakova K, Balluff B, Tressler C, Adelaja T, Heeren R, Glunde K . Cellular resolution in clinical MALDI mass spectrometry imaging: the latest advancements and current challenges. Clin Chem Lab Med. 2019; 58(6):914-929. PMC: 9867918. DOI: 10.1515/cclm-2019-0858. View

Ogrinc Potocnik N, Porta T, Becker M, Heeren R, Ellis S . Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam. Rapid Commun Mass Spectrom. 2015; 29(23):2195-203. DOI: 10.1002/rcm.7379. View

Zavalin A, Yang J, Hayden K, Vestal M, Caprioli R . Tissue protein imaging at 1 μm laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS. Anal Bioanal Chem. 2015; 407(8):2337-42. PMC: 4359058. DOI: 10.1007/s00216-015-8532-6. View

Harris W, Janecki D, Reilly J . Use of matrix clusters and trypsin autolysis fragments as mass calibrants in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2002; 16(18):1714-22. DOI: 10.1002/rcm.775. View

10.

Hoiem T, Andersen M, Martin-Lorenzo M, Longuespee R, Claes B, Nordborg A . An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue. Proteomics. 2022; 22(10):e2100223. PMC: 9285595. DOI: 10.1002/pmic.202100223. View

11.

Claes B, Krestensen K, Yagnik G, Grgic A, Kuik C, Lim M . MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined. Anal Chem. 2023; 95(4):2329-2338. PMC: 9893213. DOI: 10.1021/acs.analchem.2c04220. View

12.

Derks J, Leduc A, Wallmann G, Huffman R, Willetts M, Khan S . Increasing the throughput of sensitive proteomics by plexDIA. Nat Biotechnol. 2022; 41(1):50-59. PMC: 9839897. DOI: 10.1038/s41587-022-01389-w. View

13.

Barsnes H, Eidhammer I, Cruciani V, Mikalsen S . Protease-dependent fractional mass and peptide properties. Eur J Mass Spectrom (Chichester). 2008; 14(5):311-7. DOI: 10.1255/ejms.934. View

14.

Ucal Y, Ozpinar A . Improved spectra for MALDI MSI of peptides using ammonium phosphate monobasic in MALDI matrix. J Mass Spectrom. 2018; 53(8):635-648. DOI: 10.1002/jms.4198. View

15.

Seidler J, Zinn N, Boehm M, Lehmann W . De novo sequencing of peptides by MS/MS. Proteomics. 2009; 10(4):634-49. DOI: 10.1002/pmic.200900459. View

16.

Angel P, Norris-Caneda K, Drake R . In Situ Imaging of Tryptic Peptides by MALDI Imaging Mass Spectrometry Using Fresh-Frozen or Formalin-Fixed, Paraffin-Embedded Tissue. Curr Protoc Protein Sci. 2018; 94(1):e65. PMC: 6205905. DOI: 10.1002/cpps.65. View

17.

Beyer A, Christensen M, Walker B, Lestourgeon W . Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell. 1977; 11(1):127-38. DOI: 10.1016/0092-8674(77)90323-3. View

18.

Kompauer M, Heiles S, Spengler B . Atmospheric pressure MALDI mass spectrometry imaging of tissues and cells at 1.4-μm lateral resolution. Nat Methods. 2016; 14(1):90-96. DOI: 10.1038/nmeth.4071. View

19.

Nefedov A, Mitra I, Brasier A, Sadygov R . Examining troughs in the mass distribution of all theoretically possible tryptic peptides. J Proteome Res. 2011; 10(9):4150-7. PMC: 3184890. DOI: 10.1021/pr2003177. View

20.

Krause E, Wenschuh H, Jungblut P . The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins. Anal Chem. 1999; 71(19):4160-5. DOI: 10.1021/ac990298f. View