DOI: 10.25345/c55x25s2g
title: MassIVE MSV000099656 - Enhancing single-cell proteomics with high-sensitivity, ultrahigh-throughput narrow-bore open tubular liquid chromatography
title: Single-cell proteomics faces significant challenges in balancing sensitivity and throughput, primarily due to limitations in liquid chromatography (LC) separation. We addressed this challenge by implementing a low-loss sample injection method for narrow bore open tubular liquid chromatography (nOTLC). This approach enabled injection of ~20% of samples, marking a 295-fold improvement over conventional configurations. Our novel platform identified 3955 proteins from &lt;40 pg of HeLa digest loaded on the column at a throughput of 261 samples per day. We further demonstrated ability to enhance throughput to 720 samples per day while still identifying 2460 proteins from only ~20 pg of HeLa digest. The narrow chromatographic peaks combined with ultralow flow rates provided excellent electrospray ionization efficiency resulting in enhanced sensitivity for limited samples. The optimized nOTLC-MS system effectively balances the throughput-sensitivity trade-off in single-cell proteomics, providing a transformative platform for large-scale exploration of cellular heterogeneity with unmatched speed and sensitivity. For the nOTLC-MS/MS, HeLa digest solutions were prepared by reconstituting a 20 ug lyophilized sample in MPA and then serially diluting it to concentration of 5000 pg, 1000 pg, 200 pg, 100 pg, 50 pg, 25 pg, 5 pg, and 1 pg per 70 nL sample loop, using MPA for dilution. Due to the short length of the nOTC (10 cm), the 6-port valve must be placed close to the MS inlet. To address this requirement, we 3D-printed a custom holder that securely mounted both a manual XYZ translation stage and the 6-port valve actuator in front of the MS inlet. Additionally, a second 3D-printed holder was used to attach the nOTC to the XYZ translation stage, enabling precise adjustment of the emitter tip's position with the assistance of two DinoLite cameras. The low-loss injection process requires precise synchronization between the valve actuators and pumps. To achieve this, we employed automatic mouse-clicking software in combination with custom LIF data collection and valve control software to seamlessly control the pump and valves. The 261, 288, and 303 SPD throughput utilized a 4.25 min gradient followed by a 0.125 min wash with 50% mobile phase B and a 0.125 min equilibration. The 480 SPD used a 2.25 min gradient with 0.125 min wash and 0.125 min equilibration. 720 SPD separation had 20 replicates, 411 SPD and 480 SPD had 15 replicates, and all other nOTLC-MS experiments had 10 replicates, except for the blank at 261 SPD, which had 5 replicates. To determine the data-independent acquisition - parallel accumulation-serial fragmentation (dia-PASEF) windows, we performed nOTLC-MS using the 4.25 min gradient and the PASEF method with 200 pg HeLa digest per sample loop with a 180 s dedicated sample injection time while other configurations were kept the same. The spectra were acquired with an m/z range of 100-1250 and an IM range of 1.6-0.6 Vs/cm2. The ramp time was set to 100 ms. Singly charged ions were filtered out in the m/z-IM plane. The number of PASEF ramps was set to 7. The intensity threshold for fragmentation was 500. Active exclusion was set to 0.4 min. The PASEF datasets were processed using FragPipe to generate a library, which was used in py_diAID to generate dia-PASEF windows. In py_diAID19, the precursor m/z range was set to 300-1100 and the mobility range was set to 0.65-1.25 Vs/cm2, and the number of dia-PASEF scans was set to 7. All other parameters were kept at their default values. The dia-PASEF cycle time is approximately 0.86 s. For data analysis, we downloaded the Homo Sapiens (UP000005640) FASTA from UniProt on April 19, 2025. The PASEF data were analyzed using FragPipe v22.0 to generate a library for use in py_diAID with the following parameters: precursor and fragment mass tolerance of +/-20 ppm, strict trypsin cleavage, carbamidomethylation of cysteine as a fixed modification, oxidation of methionine as a variable modification, up to 2 missed cleavages, peptide length of 6-50 amino acids, peptide mass range of 500-5000 Da, and a maximum of 3 variable modifications per peptide. DIA-NN v2.1.021 was used to generate the in-silico spectral library and to analyze dia-PASEF data. For library generation, the protease was set to Trypsin/P, the maximum number of missed cleavages allowed was 2, and the maximum number of variable modifications was 3. N-terminal methionine excision, cysteine carbamidomethylation, methionine oxidation and N-terminal acetylation were enabled as a variable modification. The peptide length range was set to 5-50. The precursor charge range was set to 2-5. The precursor and fragment ion m/z ranges were set to 200-1800. For search, precursor FDR was set at 1%, both MS1 and MS2 accuracies were set as 15 ppm. The scan window was set to 10. Match between runs (MBR) was enabled.