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Label-free quantification
Label-free quantification
Label-free quantification
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Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantification, label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein.[1][2]

Implementation

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Label-free quantification experiment with 3 samples, 3 LC-MS files and 5 precursor ions/peptides. Intensities at the peak of the chromatographic peaks are used for quantification in this particular case. Peptides are identified via fragmentation mass spectra, and some of the precursor ions will be quantified, but not mapped to any peptide sequence.

Label-free quantification may be based on precursor signal intensity or on spectral counting. The first method is useful when applied to high precision mass spectra, such as those obtained using the new generation of time-of-flight (ToF), fourier transform ion cyclotron resonance (FTICR), or Orbitrap mass analyzers. The high-resolution power facilitates the extraction of peptide signals on the MS1 level and thus uncouples the quantification from the identification process. In contrast, spectral counting simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.

The computational framework of label free approach includes detecting peptides, matching the corresponding peptides across multiple LC-MS data, selecting discriminatory peptides.[3][4]

Intact protein expression spectrometry (IPEx) is a label-free quantification approach in mass spectrometry under development by the analytical chemistry group at the United States Food and Drug Administration Center for Food Safety and Applied Nutrition and elsewhere. Intact proteins are analyzed by an LCMS instrument, usually a quadrupole time-of-flight in profile mode, and the full protein profile is determined and quantified using data reduction software. Early results are very encouraging. In one study, two groups of treatment replicates from mammalian samples (different organisms with similar treatment histories, but not technical replicates) show dozens of low CV protein biomarkers, suggesting that IPEx is a viable technology for studying protein expression.[5]

Detecting peptides

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Typically, peptide signals are detected at the MS1 level and distinguished from chemical noise through their characteristic isotopic pattern. These patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the mono-isotopic peptide mass. The total ion current of the peptide signal is then integrated and used as a quantitative measurement of the original peptide concentration. For each detected peptide, all isotopic peaks are first found and the charge state is then assigned.

Label-free quantification may be based on precursor signal intensity and has problems due to isolation interference: in high-throughput studies, the identity of the peptide precursor ion being measured could easily be a completely different peptide with a similar m/z ratio and which elutes in a time frame overlapping with that of the former peptide. Spectral counting has problems due to the fact that the peptides are identified, thus making it necessary to run an additional MS/MS scan which takes time and therefore reduces the resolution of the experiment.

Matching corresponding peptides

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In contrast to differential labelling, every biological specimen needs to be measured separately in a label-free experiment. The extracted peptide signals are then mapped across few or multiple LC-MS measurements using their coordinates on the mass-to-charge and retention-time dimensions. Data from high mass precision instruments greatly facilitate this process and increase the certainty of matching correct peptide signals across runs.

Clearly, differential processing of biological samples makes it necessary to have a standard which can be used to adjust the results. Peptides that are not expected to change in their expression levels in different biological samples may be used for this purpose. However, not all peptides ionize well and therefore the choice of candidates should be done after an initial study which should only characterize the protein content of the biological samples that will be investigated.

Selecting discriminatory peptides

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Finally, sophisticated normalization methods are used to remove systematic artefacts in the peptide intensity values between LC-MS measurements. Then, discriminatory peptides are identified by selecting the peptides whose normalized intensities are different (e.g., p-value < 0.05) among multiple groups of samples.

In addition, newer hybrid mass spectrometers like LTQ OrbiTrap offer the possibility to acquire MS/MS peptide identifications in parallel to the high mass precision measurement of peptides on the MS1 level. This raises the computational challenge for the processing and integration of these two sources of information and has led to the development of novel promising quantification strategies.

References

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Label-free quantification

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Label-free quantification (LFQ) is a -based method in that determines the relative or absolute abundances of proteins within complex biological samples without employing isotopic, chemical, or fluorescent labels. This technique relies on direct analysis of data to infer protein levels, making it a versatile tool for comparative studies across diverse sample types, including those unsuitable for labeling approaches. The core strategies of LFQ encompass intensity-based quantification, which measures the areas or heights of peptide ion chromatographic peaks to gauge abundance changes, and spectral counting, which quantifies proteins by enumerating the tandem mass spectra uniquely assigned to them. Advanced algorithms, such as MaxLFQ, enhance accuracy by incorporating delayed normalization of intensities and maximal extraction of ratio information from shared peptides across samples, achieving high precision in -wide analyses. These methods are implemented in software tools like MaxQuant and Proteome Discoverer, which handle , normalization, and statistical validation to mitigate variability from and instrument performance. LFQ offers key advantages over label-based techniques, including lower costs, simpler workflows, and the capacity to process unlimited samples without constraints, thereby facilitating large-scale experiments. However, challenges such as handling missing values, ensuring reproducibility, and addressing limitations require robust normalization and statistical frameworks. In applications, LFQ has been instrumental in clinical for identification, elucidating disease mechanisms in cancer and neurodegenerative disorders, and profiling immune responses, providing insights into pathological protein alterations.

Overview

Definition and principles

Label-free quantification (LFQ) is a mass spectrometry-based approach used to determine the relative abundance of proteins in biological samples by analyzing intrinsic signal properties from liquid chromatography-mass spectrometry (LC-MS) data, such as intensities or the frequency of identified spectra, without the need for isotopic or chemical labeling. This method enables the comparison of protein levels across multiple samples by leveraging the natural variations in mass spectrometric signals, making it particularly suitable for large-scale proteomic studies where labeling is impractical or cost-prohibitive. The core principles of LFQ rely on the high reproducibility of LC-MS runs, where peptides from digested proteins are separated by liquid chromatography and ionized for mass analysis, allowing consistent detection and alignment of features across experiments. In contrast to labeled methods like stable isotope labeling by in (SILAC) or isobaric tags (e.g., iTRAQ), which introduce synthetic tags to enable direct multiplexing within a single run, LFQ avoids such modifications to reduce and expense, though it requires separate runs for each sample and sophisticated computational alignment to account for technical variability. At its foundation, LFQ operates within the framework of (MS/MS), where MS1 spectra provide precursor intensities for quantification, and MS2 spectra generate fragment ions for identification, ensuring that signals can be mapped to specific proteins. LFQ primarily supports relative quantification, measuring fold changes in protein abundance between conditions (e.g., diseased vs. healthy samples), rather than absolute quantification, which typically requires spiked internal standards. This relative approach plays a central role in differential expression analysis, facilitating the identification of biologically significant changes by integrating peptide-level data to infer protein-level ratios, often through methods like spectral counting (number of MS2 spectra per protein) or intensity-based integration of MS1 peaks. While absolute quantification can be approximated in LFQ using empirical models, its strength lies in scalable, label-free comparisons for hypothesis-driven .

Historical development

Label-free quantification (LFQ) in proteomics emerged in the early 2000s, coinciding with significant improvements in the reproducibility of liquid chromatography-mass spectrometry (LC-MS) workflows, which enabled more reliable comparison of peptide signals across multiple samples without the need for isotopic labeling. Prior to 2003, foundational efforts focused on peak matching techniques, where relative protein abundances were estimated by aligning and comparing chromatographic peak intensities or areas for the same peptides in different runs. A key early demonstration came from Bondarenko et al. in 2002, who used enzymatic digestion followed by capillary reversed-phase LC-tandem MS to identify and relatively quantify proteins in complex mixtures by directly measuring peptide ion current profiles. A major milestone occurred in 2004 with the introduction of spectral counting as a practical LFQ approach, where protein abundance is approximated by the number of MS/MS spectra assigned to each protein, offering a simple, label-free proxy for relative quantification. et al. developed a linking spectral counts to protein abundance levels in label-free LC-MS experiments, validating its utility on standard mixtures and complex samples like lysates. By the mid-2000s, the field shifted toward intensity-based methods, which measure precursor intensities or peak areas in MS1 scans for greater sensitivity and accuracy. Old et al. in 2005 compared spectral counting with intensity measurements in , showing that intensity-based approaches better captured abundance changes in cell line digests, though both required careful normalization to account for run-to-run variations. This period also saw widespread adoption of LFQ in studies around 2006, as evidenced by its integration into routine differential expression analyses in diverse biological systems. Technological advancements in high-resolution mass spectrometry, such as the commercial introduction of the Orbitrap analyzer in 2005, further propelled LFQ by providing the mass accuracy and resolution needed for precise peptide feature detection and alignment across samples. Computational tools also evolved to handle the growing data complexity, facilitating automated peak extraction and normalization. In the 2010s, LFQ integrated with data-independent acquisition (DIA) strategies, enhancing comprehensiveness and reproducibility; for instance, SWATH-MS in 2012 enabled targeted, label-free quantification of thousands of proteins in a discovery mode. Labeled alternatives like SILAC, introduced around 2002, provided complementary options but LFQ gained traction for its cost-effectiveness and flexibility in large-scale studies. In the , LFQ has seen enhancements tailored to challenging samples like human plasma, with multicenter evaluations demonstrating improved precision and depth through optimized workflows and advanced instrumentation. These developments have solidified LFQ as a cornerstone of , supporting applications from discovery to .

Quantification Methods

Spectral counting

Spectral counting is a straightforward label-free quantification technique in mass spectrometry-based proteomics that estimates protein relative abundance by tallying the number of tandem mass spectrometry (MS/MS) spectra matched to peptides from each protein. This method operates under the assumption that higher-abundance proteins produce a greater number of detectable peptide ions, resulting in proportionally more identifiable MS/MS fragments during data-dependent acquisition. Introduced as a practical surrogate for protein levels in shotgun proteomics workflows, spectral counting leverages the stochastic sampling inherent to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to infer abundance without requiring isotopic labeling. To mitigate run-to-run variations in total ion current or acquisition efficiency, spectral counts are commonly normalized by dividing each protein's count by the total number of spectra observed in the sample, yielding a normalized spectral abundance factor that facilitates comparative analysis across experiments. For absolute quantification estimates, the exponentially modified protein abundance index (emPAI) refines this approach by accounting for protein-specific peptide observability. The emPAI is calculated as follows: emPAI=10Nobserved/Ndetectable−1\text{emPAI} = 10^{N_{\text{observed}} / N_{\text{detectable}}} - 1 where NobservedN_{\text{observed}} represents the number of unique peptides sequenced for the protein, and NdetectableN_{\text{detectable}} denotes the theoretical number of observable peptides based on the protein's sequence and protease digestion. This index correlates linearly with protein molar content, enabling broader applicability in diverse proteomic datasets. The primary advantage of spectral counting lies in its operational simplicity, as it bypasses the complexities of chromatographic peak detection and alignment required in other label-free methods, rendering it well-suited for standard data-dependent acquisition protocols in discovery . This count-based metric provides robust relative quantification over a spanning approximately two orders of magnitude, with correlations to protein amounts validated in complex mixtures like cell lysates. Despite its ease of implementation, counting exhibits limitations in sensitivity for low-abundance proteins, where incomplete and sampling can lead to underestimation or missed detections due to identification variability across replicates. This arises from the random selection of precursor ions for fragmentation in crowded spaces, potentially biasing results toward higher-abundance and reducing for trace-level analytes. Intensity-based methods can serve as a complementary to enhance precision in such scenarios.

Intensity-based approaches

Intensity-based approaches in label-free quantification (LFQ) rely on the direct measurement of signal intensities from to infer protein abundances across samples. The core mechanism involves extracting and integrating intensities from MS1 spectra or constructing extracted ion chromatograms (XICs) from MS2 , where chromatographic peaks corresponding to are detected as features. These peaks represent the summed currents over retention time, providing an analog measure of abundance that requires prior feature detection algorithms to identify and quantify reliable signals. Common variants include the top-N method, which aggregates the intensities of the N most intense peptides (typically N=3 to 10) unique to each protein to estimate overall protein levels, reducing noise from less reliable lower-intensity signals. Another key variant employs total ion current (TIC) normalization, where peptide or protein intensities are scaled by the total ion flux across the entire chromatogram to account for technical variations in sample loading or instrument sensitivity between runs. Mathematically, relative protein abundance ratios between samples are computed as the normalized intensity in sample 1 divided by the normalized intensity in sample 2, often expressed as: Ratio=Isample1/Nfactor1Isample2/Nfactor2\text{Ratio} = \frac{I_{sample1} / N_{factor1}}{I_{sample2} / N_{factor2}} where II denotes the summed intensity and NfactorN_{factor} is the normalization factor (e.g., ). These ratios are typically log2-transformed to stabilize variance and facilitate statistical , yielding values centered around 0 for equal abundances. These methods excel in quantitative accuracy, particularly in (DIA) modes where comprehensive fragmentation enables robust XIC reconstruction, and they capture a broader —up to five orders of magnitude—compared to spectral counting, which is better suited for rough abundance estimates in data-dependent acquisition (DDA).

Data Processing Workflow

Peptide detection

In (LFQ) workflows for , detection initiates the data processing pipeline by identifying and extracting signals from raw liquid chromatography-tandem (LC-MS/MS) data. This process primarily relies on database searching, where experimental MS/MS spectra are matched against theoretical fragment spectra derived from a , enabling the assignment of sequences to observed ions. Seminal search engines such as SEQUEST, which correlates spectra using scores, and , which employs probabilistic scoring based on and MS/MS data, are foundational tools for this identification step. De novo sequencing complements database methods by computationally reconstructing sequences directly from MS/MS fragmentation patterns, particularly useful for novel or post-translationally modified peptides not represented in standard databases; algorithms such as those in PEAKS, which use novel scoring models to interpret MS/MS fragmentation patterns and generate high-confidence sequences. Following spectral matching, feature detection extracts chromatographic peaks corresponding to identified peptides from the MS1 level of the LC-MS data. This involves preprocessing steps such as centroiding, which converts continuous profile-mode spectra into discrete peak lists by fitting Gaussian models to signals, and noise filtering, often using transforms or intensity thresholds to suppress chemical and electronic while preserving true peptide envelopes. Advanced open-source tools like refine this by integrating isotope detection and charge state deconvolution, achieving higher sensitivity for low-abundance features in complex samples compared to earlier methods. To ensure reliability, peptide identifications are subjected to (FDR) control, typically at a stringent 1% threshold, using the target-decoy approach where reverse-sequence decoys estimate false positives among target hits. The detection strategy varies by acquisition mode: in data-dependent acquisition (DDA), the instrument dynamically selects the top N most intense precursor ions (e.g., N=10–20) from each MS1 scan for targeted fragmentation, prioritizing abundant peptides but introducing variability and potential undersampling of low-abundance ones across replicate runs. In (DIA), all precursor ions within systematic isolation windows (e.g., 25–100 m/z units) are fragmented concurrently, generating multiplexed MS/MS spectra that require spectral library-assisted for extraction, thus enhancing in LFQ. Recent innovations, such as narrow-window DIA (nDIA), further enhance these capabilities by enabling ultra-fast MS/MS scans for deeper coverage. During detection, missing features—arising from stochastic ionization or below-detection-limit signals—are addressed with stage-specific imputation, such as deterministic left-censored methods that replace absences with a fixed fraction of the minimum observed intensity, tailored to the random or censored nature of early omissions. These detected features form the basis for subsequent alignment across samples to enable quantitative comparisons.

Peptide alignment and matching

Peptide alignment and matching in label-free quantification (LFQ) involves mapping detected features across multiple liquid chromatography-mass spectrometry (LC-MS) runs to enable comparative analysis of their abundances, compensating for technical variations inherent to separate sample processing. This step is essential because LFQ lacks isotopic labels for , leading to run-to-run differences in that must be corrected for accurate cross-sample quantification. Alignment techniques primarily focus on retention time (RT) normalization and mass-to-charge (m/z) tolerance matching to align elution profiles. RT normalization corrects shifts in elution times using methods such as , which applies least-squares fitting to estimate global deviations between runs, or locally estimated scatterplot smoothing (), a non-linear approach that handles segment-specific drifts by fitting smoothed curves to RT deviations. often performs well for minor, systematic shifts, while is preferred for non-linear variations common in longer gradients or complex samples. m/z tolerance matching ensures features are linked within a predefined window (typically 5-20 ppm), accounting for instrument accuracy to pair ions with similar precursor masses after RT alignment. Matching strategies employ vector alignment or landmark-based methods to handle chromatographic variability, such as inconsistencies or column degradation. In landmark-based approaches, high-confidence peptides (landmarks) are manually or automatically selected from a reference run—often a pooled sample—and used to guide the alignment of other runs by establishing correspondence points. Vector alignment extends this by connecting landmarks across runs with vectors that define a warping function, iteratively refining the RT scale to minimize deviations for all features. These strategies address run-to-run variability by propagating alignments from reliable landmarks to less certain features, improving overall map completeness. Specific algorithms, such as the warping function in Progenesis QI, automate this process by selecting an optimal reference run and using alignment vectors derived from ions to non-linearly transform RT axes, achieving precise overlay of chromatograms. Match acceptance criteria typically involve a composite score combining RT alignment quality, m/z proximity, and intensity similarity, with thresholds set to control false discovery rates (FDR), such as requiring scores above a user-defined or FDR <1% for transferred identifications. Advanced models, like Bayesian Dirichlet process Gaussian mixture models, further incorporate ion mobility or product ion data to enhance matching confidence without rigid distance cutoffs. Key challenges in peptide alignment for LFQ include RT drift, which can be nonlinear and exceed 5-10% of gradient length due to factors like temperature fluctuations or mobile phase variations, complicating feature correspondence in datasets with thousands of peptides. This issue is exacerbated in LFQ by the absence of labels, which prevents direct multiplexing and amplifies the need for robust post-acquisition corrections to avoid quantification biases.

Protein-level quantification

Protein-level quantification in label-free proteomics aggregates peptide-level measurements, typically derived from aligned and matched peptides across samples, to infer overall protein abundances. Common aggregation methods include summing the intensities or spectral counts of associated peptides, which provides a direct measure of total signal, or computing the median to mitigate the effects of outlier peptides with unusually high or low signals. Summing is particularly suited for spectral counting approaches, where the total number of spectra per protein correlates with abundance, while median normalization is favored in intensity-based methods to enhance robustness against technical variability. These strategies assume that peptide signals are proportional to protein concentration, though they require careful handling of missing values and variable peptide coverage. Shared peptides, which map to multiple proteins due to sequence homology or isoforms, complicate aggregation and are addressed through parsimony analysis. This approach employs bipartite graph modeling to parsimoniously assign peptides to the minimal set of proteins that explain all identifications, improving accuracy and reducing redundancy in protein inference. By resolving ambiguities, parsimony ensures that shared signals are distributed without over- or under-representing proteins, as demonstrated in analyses of complex mixtures where it increased identification transparency and precision compared to simple exclusion methods. High-confidence assignments typically incorporate false discovery rate (FDR) thresholds below 1% at the peptide level to filter unreliable matches. To further refine quantification, discriminatory peptide selection prioritizes unique, high-confidence peptides exclusive to a single protein, using criteria such as low intensity variance across replicates, high signal-to-noise ratios, and absence of post-translational modifications that could skew measurements. This selection excludes shared or low-abundance peptides, focusing on proteotypic ones that serve as reliable surrogates for the protein, thereby enhancing specificity and reducing noise in downstream analyses. For instance, tools like MaxQuant implement filters for peptide exclusivity and confidence scores to curate these sets automatically. Normalization at the protein level often employs intensity-based absolute quantification (iBAQ) as an extension of label-free methods, where protein abundance is estimated by dividing the sum of all observed peptide intensities by the number of theoretically observable tryptic peptides (typically 6-40 residues long) for that protein:
iBAQ=∑peptide intensitiesnumber of theoretical peptides\text{iBAQ} = \frac{\sum \text{peptide intensities}}{\text{number of theoretical peptides}}
This yields copy numbers per cell proportional to absolute abundance, correlating strongly with independent validations over four orders of magnitude and outperforming relative methods in precision for cross-sample comparisons. iBAQ assumes complete tryptic digestion and uniform ionization efficiency, making it particularly useful for estimating stoichiometry in cellular proteomes. Following aggregation and normalization, statistical validation assesses the significance of protein-level ratios or abundances between conditions using parametric tests like the Student's t-test for two-group comparisons or analysis of variance (ANOVA) for multi-group designs. These tests evaluate differential expression by modeling variance from technical and biological replicates, often after log-transformation to stabilize variances, and incorporate multiple-testing corrections (e.g., Benjamini-Hochberg) to control false positives. Such approaches have been benchmarked to achieve high sensitivity in detecting fold-changes as low as 1.5 in label-free datasets, provided sufficient replicates (n ≥ 3) are included.

Software Tools

Open-source tools

Open-source tools for label-free quantification (LFQ) in proteomics provide accessible, modifiable platforms that enable researchers to process mass spectrometry data without licensing fees, often integrating search engines, quantification algorithms, and workflow customization. These tools support various acquisition modes and data formats, facilitating both academic and collaborative research environments. MaxQuant, introduced in 2008, revolutionized LFQ accessibility by offering a comprehensive software suite for data-dependent acquisition (DDA) and data-independent acquisition (DIA) workflows. It incorporates the Andromeda search engine for peptide identification and a dedicated LFQ module that computes protein intensities based on peptide precursor ion signals, enabling the analysis of up to thousands of samples in a single run. MaxQuant directly processes raw files from major vendors like Thermo Fisher Scientific and Bruker, supporting relative quantification across large-scale experiments such as proteome-wide studies. OpenMS serves as a versatile open-source C++ library and toolkit for building custom LFQ pipelines, emphasizing modular components for data management, analysis, and visualization. Key features include the FeatureFinder for detecting chromatographic peaks in MS1 spectra and alignment tools like MapAlignerPoseidon for matching features across samples to enable accurate intensity-based quantification. With Python bindings via pyOpenMS, users can integrate it into scripting environments for tailored workflows, supporting label-free approaches in diverse applications from standard bottom-up proteomics to metabolomics. Skyline, an open-source Windows application, excels in both targeted and untargeted LFQ, particularly through its robust extraction of extracted ion chromatograms (XICs) from MS1 data for peptide-level quantification. It facilitates label-free analysis via MS1 filtering, allowing users to build methods for selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and full-scan data, with strong visualization tools for peak integration and quality assessment. Community-driven development ensures frequent updates, making it suitable for interactive exploration of LFQ datasets in proteomics research. AlphaPept, introduced in 2024, is a Python-based open-source framework designed for efficient processing of large high-resolution mass spectrometry datasets. It supports LFQ through modular feature extraction and parallelized analysis, enabling rapid quantification in DDA workflows with high sensitivity and speed. DIA-NN is an open-source tool specialized for data-independent acquisition (DIA) proteomics, providing accurate LFQ via deep neural networks for peptide detection and quantification. It handles complex DIA data efficiently, supporting large-scale studies with robust normalization and imputation strategies.

Commercial software

Commercial software for label-free quantification (LFQ) in proteomics provides user-friendly interfaces, robust support, and seamless integration with vendor-specific mass spectrometry hardware, making it suitable for laboratories prioritizing workflow efficiency over customizability. These tools often include proprietary algorithms for automated data processing, statistical analysis, and reporting, with licensing models frequently bundled with instrument purchases to facilitate adoption in commercial settings. Progenesis QI, developed by Nonlinear Dynamics and now distributed by Waters Corporation, offers automated alignment of LC-MS runs using vector-based methods to correct retention time shifts, a key advancement introduced in the 2010s for improving quantification accuracy in label-free workflows. It excels in data-independent acquisition (DIA) analysis, supporting Waters-specific formats like MSE and HDMSE, and includes built-in statistical tools for identifying significantly changing proteins across large sample sets. The software integrates directly with Waters instrumentation, leveraging ion mobility data for enhanced precision, and its pricing is often tied to hardware bundles, providing perpetual licenses with maintenance options. Scaffold LFQ, from Proteome Software, emphasizes protein inference and validation through probabilistic scoring and false discovery rate control, enabling reliable assembly of peptide identifications into proteins for LFQ experiments. It supports quantification via spectral counting or precursor ion intensities, with integrated statistical tests for differential expression and visualizations like (PCA) and heatmaps. For post-translational modifications (PTMs), the companion Scaffold PTM module allows modsite-level analysis and export of quantitative data in formats compatible with external statistical software such as . Pricing follows a one-time model, with academic rates at $9,995 and commercial at $12,995, independent of hardware vendors but offering short-term options for flexibility. Thermo Fisher Scientific's Proteome Discoverer features modular nodes for LFQ, including the Minora feature detector for untargeted peak identification and quantification in MS1 spectra, optimized for label-free workflows without requiring prior identifications. It provides seamless integration with Orbitrap mass spectrometers, supporting diverse acquisition modes like data-dependent acquisition (DDA) and DIA, and allows customizable pipelines for peptide-to-protein summarization. Licensing includes base versions with upgrades (e.g., CHIMERYS subscriptions starting at annual fees) often bundled with Thermo instruments to streamline proteomics analysis in vendor ecosystems. In contrast to open-source alternatives, commercial tools like these offer dedicated and validated integrations, though at a higher cost for non-academic users.

Advantages and Limitations

Key advantages

Label-free quantification (LFQ) in mass spectrometry-based offers significant cost-effectiveness by eliminating the need for expensive reagents, such as those required in stable isotope labeling by in (SILAC) or tandem mass tag (TMT) methods. This approach reduces per-sample expenses and enables the analysis of unlimited numbers of samples through sequential liquid chromatography-mass spectrometry (LC-MS) runs, making it particularly suitable for large-scale studies or cohorts with limited budgets. The simplicity and flexibility of LFQ further enhance its utility, as it avoids biases introduced by labeling processes, including incomplete incorporation of labels or differential effects that can skew quantitative results. Unlike labeling methods that are restricted to specific biological systems (e.g., cell cultures for SILAC), LFQ is applicable to any sample without prior sequence knowledge or modifications, streamlining and broadening its experimental scope. LFQ provides a broad , leveraging the inherent sensitivity of to detect and quantify proteins across low- to high-abundance levels in complex samples, which supports comprehensive coverage in discovery workflows. Additionally, by circumventing , LFQ reduces ratio distortions arising from isotope effects on or chromatographic , ensuring more accurate relative abundance measurements. This foundation also facilitates extensions to absolute quantification, such as the intensity-based absolute quantification (iBAQ) method, which estimates protein copy numbers by normalizing total intensities to the number of tryptic peptides.

Challenges and limitations

Label-free quantification (LFQ) in faces significant reproducibility challenges due to its sensitivity to variations in liquid chromatography (LC) gradients and (MS) noise, which can lead to inconsistent elution times and signal intensities across runs. For instance, retention time shifts of approximately 3 minutes have been observed in repeated injections, necessitating precise alignment algorithms to maintain comparability. Additionally, sampling in data-dependent acquisition (DDA) modes contributes to variable identification, with only a small fraction (e.g., 0.8%) of peptides consistently detected across multiple injections of the same sample. Quantitative precision in LFQ is particularly limited for low-abundance proteins, where detection thresholds result in high rates of missing values (10-50% overall, up to 70-90% across samples), complicating accurate measurement. Imputation strategies for these missing values, such as mean imputation, often introduce by distorting data distributions, while more advanced methods like can still overestimate uncertainty when missingness exceeds 30% per protein. This reduces the reliability of LFQ for subtle biological changes, with coefficients of variation (CVs) reaching up to 63% for certain peptides. The large volume of data generated in LFQ experiments poses substantial computational demands, especially for datasets exceeding 100 samples, where processing times and memory requirements can overwhelm standard workstations. Public repositories like host over 42,000 datasets as of August 2024, with large-scale LFQ analyses (e.g., >1,000 MS runs) requiring specialized parallelization to handle the stochastic inconsistencies from DDA sampling. Recent tools such as quantms, a cloud-based pipeline, address these demands by enabling faster reanalysis of public . Unique challenges in LFQ include run-to-run carryover effects, which cause random fluctuations in peptide abundances and degrade precision, as well as inherently lower accuracy compared to labeled methods for detecting subtle changes below 2-fold. For example, LFQ detects only about 30% of 1.5-fold changes at a 5% false discovery rate, versus over 80% with tandem mass tag (TMT) approaches, due to higher variability and missing data.

Applications

Proteomics research

Label-free quantification (LFQ) has become integral to discovery in fundamental by enabling differential analysis of protein abundances across biological conditions in model organisms. In studies, LFQ has revealed proteome-wide changes during stress responses, such as heat shock in , where chaperones like show increased abundance to maintain . Similarly, LFQ-based comparative has identified early signaling events in exposed to environmental stressors, highlighting differentially regulated proteins in pathways like biosynthesis and . In protein interaction mapping, LFQ facilitates the quantification of protein complexes without isotopic labeling, allowing for the assessment of interaction stoichiometries and dynamics. This approach integrates seamlessly with protein-protein interaction (PPI) networks, as demonstrated in affinity purification-mass spectrometry workflows where LFQ scores interaction confidence using tools like SAINT, enabling the construction of high-fidelity interactomes in human and yeast systems. By comparing bait-prey abundance ratios, LFQ helps distinguish stable complexes from transient associations, providing quantitative insights into network topology. For post-translational modifications (PTMs), LFQ supports site-specific quantification in signaling pathways, capturing dynamic changes in or ubiquitination without labeling artifacts. In phosphoproteomics of signaling cascades, LFQ has quantified thousands of sites in response to stimuli like growth factors, revealing pathway-specific in model cell lines. Tools like FLEXIQuant-LF extend this to measure modification occupancy, aiding the study of kinase-substrate relationships in cellular . LFQ is widely applied in for proteome-wide profiling, where it processes complex peptide mixtures to quantify thousands of proteins in a single run, as evaluated in discovery workflows emphasizing and depth. Advances in the 2020s have extended LFQ to single-cell proteomics, achieving coverage of over 1,000 proteins per cell using on high-resolution mass spectrometers in mammalian cells, while enabling heterogeneity analysis in microbial populations at the strain level.

Biomedical and clinical uses

Label-free quantification (LFQ) has been instrumental in cancer for comparing tumor and normal tissues, enabling the identification of differentially expressed proteins that serve as biomarkers for early detection and prognosis. In , LFQ-based plasma revealed aberrant expression of proteins such as (FN), leucine-rich alpha-2-glycoprotein (LRG), complement C9 (C9), alpha-1-antitrypsin (A1AT), and alpha-1-acid glycoprotein 1 (AGP1), distinguishing profiles from healthy controls with high specificity. Studies from the 2010s onward, such as those using LFQ with nano-LC-MS/MS, identified biomarkers like and HSP90β in lung adenocarcinoma, and actinin-4, annexin A2, and IGFBP2 in , facilitating non-invasive liquid biopsies for early . These applications underscore LFQ's role in translational , where plasma-based analyses support the development of diagnostic panels without . In drug response monitoring, LFQ quantifies pharmacodynamic changes to assess therapeutic efficacy and personalize medicine. The solvent proteome profiling in cells (SPICE) method, a label-free biophysical approach, monitors protein-drug interactions in live cells by detecting stability shifts, identifying targets like (DHFR) for and (BTK) for , while revealing off-target effects. This enables proteome-wide deconvolution of drug mechanisms, with SPICE complementing thermal shift assays by quantifying over 5,000 proteins and supporting tailored therapies in . LFQ has also pinpointed predictive biomarkers for chemotherapy response in , such as (GSN), 1 (CALM1), and (TXN), which correlate with treatment outcomes and inform patient stratification. For infectious diseases, LFQ elucidates pathogen-host interactions by mapping shifts in clinical samples. In , longitudinal plasma LFQ analysis of over 1,100 hospitalized patients detected nucleoprotein with increasing frequency in severe cases (up to 41% in critical groups), alongside host responses involving , complement activation, and degradation. This revealed 305 severity-associated proteins, including (ELN) and IL1 receptor-like 1 (IL1RL1), forming prognostic panels with AUROC values of 0.85 for mortality prediction, aiding and outcome forecasting. Such applications extend to , where LFQ profiled abundance in formulations, informing and safety assessments. Multicenter plasma studies have advanced LFQ for clinical reliability, with a 2025 across 12 sites using a human plasma benchmark spiked with and E. coli proteins demonstrating (DIA) superiority over data-dependent acquisition (DDA), achieving 84.2% protein consistency, average identification of 3,193 groups, and (CV) below 6%. These efforts highlight LFQ's potential for reproducible discovery in diagnostics. Integration with enhances pattern recognition in LFQ data, where models like DIA-NN improve quantification in clinical cohorts, distilling multi-analyte panels for diseases like Alzheimer's with superior accuracy over traditional methods.

Comparison to Labeled Methods

Fundamental differences

Label-free quantification (LFQ) in mass spectrometry-based proteomics differs fundamentally from labeled methods such as stable isotope labeling by in (SILAC) and tandem mass tag (TMT) approaches in its reliance on the absence of isotopic or chemical labels. LFQ analyzes samples in sequential, independent liquid chromatography-mass spectrometry (LC-MS) runs, measuring inherent signals like intensities at the MS1 level or spectral counts, without introducing exogenous labels that alter molecular masses. In contrast, SILAC incorporates stable heavy isotopes (e.g., 13C and 15N) metabolically into proteins during , creating mass shifts observable in MS1 spectra for relative quantification upon mixing samples early in the workflow. TMT employs isobaric chemical tags attached to s post-digestion, which release reporter s during MS2 fragmentation for multiplexed quantification, allowing co-analysis of multiple samples in a single run but introducing tag-specific fragmentation patterns. This label-free strategy circumvents issues like incomplete labeling efficiency in SILAC, which typically achieves 90-95% incorporation and can bias ratios if not fully realized. A core distinction lies in sample throughput and multiplexing capabilities. LFQ supports unlimited sample numbers through separate runs, offering flexibility for diverse or large cohorts without channel constraints, though it requires stable across analyses to ensure comparability. Labeled methods, however, are limited by the number of available isotopic variants or tag channels; SILAC commonly uses 2-6 plex configurations due to metabolic constraints in , while TMT enables up to 18-plex with recent reagents like TMTpro, reducing overall run time but capping simultaneous samples per experiment. This multiplexing in labeled approaches minimizes technical variability from run-to-run differences but demands compatible sample types (e.g., cultured cells for SILAC) and can introduce batch effects across multiple labeling sets. Precision and quantitative accuracy also vary significantly between LFQ and labeled techniques. LFQ typically exhibits higher coefficients of variation (CVs) of 20-30% for protein abundances, stemming from inter-run variability in and detection, though it avoids isotope dilution effects that can subtly skew ratios in labeled methods by altering populations. Labeled quantification generally achieves lower CVs around 10%, benefiting from co-elution and internal normalization in multiplexed setups; for instance, SILAC's early mixing reduces artifacts, while TMT's reporter ions provide precise but suffer from compression due to co-isolated impurities. These trade-offs highlight LFQ's strength in unbiased signal versus the enhanced of labeled strategies at the cost of labeling artifacts.

Method selection guidelines

Label-free quantification (LFQ) is particularly advantageous for experiments involving large sample cohorts, such as those exceeding 50 samples, due to its scalability and ability to handle extensive datasets without the multiplexing limitations of labeled approaches. This makes LFQ ideal for clinical or epidemiological studies requiring analysis of numerous biological replicates, where labeled methods like tandem mass tags (TMT) may become logistically challenging despite their multiplexing capacity. Conversely, labeled methods are better suited for smaller, high-precision datasets, such as targeted validation experiments with fewer than 20 samples, where isotopic labeling ensures consistent quantification across runs. Budget constraints further favor LFQ, as it eliminates the costs associated with isotopic reagents and labeling kits, which can significantly increase expenses in labeled workflows. In terms of sample types, LFQ excels with non-culturable or complex biological materials, such as clinical tissues, biofluids, or environmental samples, where metabolic labeling is infeasible. For instance, LFQ enables direct quantification from heterogeneous tissue extracts without prior requirements, preserving native states. Labeled methods, particularly stable isotope labeling by in (SILAC), are more appropriate for homogeneous, culturable samples like cell lines, where in vivo incorporation of heavy provides high-fidelity relative quantification. Regarding fold-change sensitivity, labeled approaches demonstrate superior performance for detecting subtle protein abundance variations below 1.5-fold, owing to their internal standards that minimize technical variability. LFQ, while capable of quantifying broader dynamic ranges (e.g., >2-fold changes), often requires orthogonal validation techniques, such as targeted assays, to confirm low-magnitude differences due to potential run-to-run inconsistencies. For workflow integration, LFQ pairs effectively with (DIA) modes to enhance consistency and reproducibility across large-scale analyses, as DIA's comprehensive fragmentation reduces missing values inherent in data-dependent acquisition (DDA). Recent 2023 reviews advocate hybrid LFQ-DIA pipelines for untargeted , recommending them for studies needing both depth and throughput, while suggesting labeled-DIA combinations only for scenarios demanding isotopic precision.

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