Comprehensive, reproducible and precise analysis of large sample cohorts is one of the key objectives of quantitative proteomics. Here, we present an implementation of data-independent acquisition using its parallel acquisition nature that surpasses the limitation of serial MS2 acquisition of data-dependent acquisition on a quadrupole ultra-high field Orbitrap mass spectrometer. In deep single shot data-independent acquisition, we identified and quantified 6,383 proteins in human cell lines using 2-or-more peptides/protein and over 7100 proteins when including the 717 proteins that were identified on the basis of a single peptide sequence. 7739 proteins were identified in mouse tissues using 2-or-more peptides/protein and 8121 when including the 382 proteins that were identified based on a single peptide sequence. Missing values for proteins were within 0.3 to 2.1% and median coefficients of variation of 4.7 to 6.2% among technical triplicates. In very complex mixtures, we could quantify 10,780 proteins and 12,192 proteins when including the 1412 proteins that were identified based on a single peptide sequence. Using this optimized DIA, we investigated large-protein networks before and after the critical period for whisker experience-induced synaptic strength in the murine somatosensory cortex 1-barrel field. This work shows that parallel mass spectrometry enables proteome profiling for discovery with high coverage, reproducibility, precision and scalability.

The properties of the 2,570 Chinese characters explicitly taught in Chinese elementary schools were systematically investigated, including types of characters, visual complexity, spatial structure, phonetic regularity and consistency, semantic transparency, independent and bound components, and phonetic and semantic families. Among the findings are that the visual complexity, phonetic regularity, and semantic transparency of the Chinese characters taught in elementary school increase from the early grades to the later grades: Characters introduced in the 1st or 2nd grade typically contain fewer strokes, but are less likely to be regular or transparent, than characters introduced in the 5th or 6th grade. The inverse relation holds when characters are stratified by frequency. Low-frequency characters tend to be visually complex, phonetically regular, and semantically transparent whereas high-frequency characters tend to be the opposite. Combined with other findings, the analysis suggests that written Chinese has a logic that children can understand and use.

Here we describe the use of data independent acquisition (DIA) on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment (freely available on-line at http://skyline.maccosslab.org). The systematic acquisition of MS/MS spectra by DIA is in contrast to DDA where the acquired MS/MS spectra are only suitable for identification of a stochastically sampled set of peptides. Similar to selected reaction monitoring (SRM), peptides can be quantified from DIA data using targeted chromatogram extraction. Unlike SRM, data acquisition is not constrained to a pre-determined set of target peptides. In this protocol, a spectral library is generated using data dependent acquisition (DDA), and chromatograms are extracted from the DIA data for all peptides in the library. Similar to SRM, quantification using DIA data is based on the area under the curve of extracted MS/MS chromatograms. Additionally, a quality control method suitable for DIA based on targeted MS/MS acquisition is detailed. Not including time spent acquiring data, and time for database searching, the procedure takes about 1–2 hours to complete. Typically, data acquisition requires roughly 1–4 hours per sample and a database search will take 0.5–2 hours to complete.

We have applied high-field asymmetric waveform ion mobility spectrometry (FAIMS) to the analysis of the phosphopeptides APLpSFRGSLPKSYVK, APLSFRGpSLPKSYVK, and APLSFRGSLPKpSYVK. The peptides have identical amino acid sequences and differ only in the site of phosphorylation. The results show that FAIMS is capable of at least partially separating these species. Separation was confirmed by coupling FAIMS with high-resolution electron transfer dissociation (ETD) mass spectrometry. Phosphorylation is retained on the ETD peptide fragments thereby allowing assignment of the site of the modification. Co-eluting phosphopeptides which differ only in the site of modification are frequently observed in liquid chromatography/tandem mass spectrometry phosphoproteomics experiments, and therefore these proof-of-principle results have implications for the application of FAIMS in that field.

The promises of data-independent acquisition (DIA) strategies are a comprehensive and reproducible digital qualitative and quantitative record of the proteins present in a sample. We developed a fast and robust DIA method for comprehensive mapping of the urinary proteome that enables large scale urine proteomics studies. Compared to a data-dependent acquisition (DDA) experiments, our DIA assay doubled the number of identified peptides and proteins per sample at half the coefficients of variation observed for DDA data (DIA = ~8%; DDA = ~16%). We also tested different spectral libraries and their effects on overall protein and peptide identifications and their reproducibilities, which provided clear evidence that sample type-specific spectral libraries are preferred for reliable data analysis. To show applicability for biomarker discovery experiments, we analyzed a sample set of 87 urine samples from children seen in the emergency department with abdominal pain. The whole set was analyzed with high proteome coverage (~1300 proteins/sample) in less than 4 days. The data set revealed excellent biomarker candidates for ovarian cyst and urinary tract infection. The improved throughput and quantitative performance of our optimized DIA workflow allow for the efficient simultaneous discovery and verification of biomarker candidates without the requirement for an early bias toward selected proteins.