Low Molecular Weight Serum Proteome Research Articles

Improved profiling of low molecular weight serum proteome for gastric carcinoma by data-independent acquisition.

Low molecular weight proteins (LMWPs) in the bloodstream participate in various biological processes and are closely associated with disease status, whereas identification of serous LMWPs remains a great technical challenge due to the wide dynamic range of protein components. In this study, we constructed an integrated LMWP library by combining the LMWPs obtained by three enrichment methods (50% ACN, 20% ACN + 20mM ABC, and 30kDa) and their fractions identified by the data-dependent acquisition method. With this newly constructed library, we comprehensively profiled LMWPs in serum using data-independent acquisition and reliably achieved quantitative results for 75% serous LMWPs. When applying this strategy to quantify LMWPs in human serum samples, we could identify 405 proteins on average per sample, of which 136 proteins were with a MW less than 30kDa and 293 proteins were with a MW less than 65kDa. Of note, pre- and post-operative gastric carcinoma (GC) patients showed differentially expressed serous LWMPs, which was also different from the pattern of LWMP expression in healthy controls. In conclusion, our results showed that LMWPs could efficiently distinguish GC patients from healthy controls as well as between pre- and post-operative statuses, and more importantly, our newly developed LMWP profiling platform could be used to discover candidate LMWP biomarkers for disease diagnosis and status monitoring.

High Resolution Continuous Elution Electrophoresis for the Evaluation of Low Abundance Serum Proteins.

The evaluation of low abundance biomarkers in the circulating low molecular weight serum proteome is an important source of information. Techniques for sample preparation to remove high abundant proteins and to enrich the low molecular weight fraction are usually required prior to novel biomarker detection. A continuous elution electrophoresis was used to separate the low molecular weight serum proteins from the high abundance serum proteins, such as albumin and immunoglobulins. Centrifugal concentration, SDS-PAGE, and total protein staining were performed to analyze eluted protein fractions. Consecutive concentrated serum protein fractions demonstrate separation at a high resolution of 1 - 2 kDa below 20 kDa. Continuous elution electrophoresis is an adequate method to eliminate high abundance proteins which interfere with the detection of low abundance biomarkers in the low molecular weight proteome and to enrich its proteins for subsequent detection and clinical evaluation.

Preparation of the Low Molecular Weight Serum Proteome for Mass Spectrometry Analysis.

The ability to cure or manage many diseases is highly dependent on the ability to correctly diagnose them at the earliest possible stage. Diagnosis relies heavily on biomarkers whether these be visual symptoms or molecules found within samples acquired from the patient. For conditions that lack useful biomarkers, researchers are often faced with the task of sifting through very complex biological samples (i.e., serum, plasma, urine, tissue, cells, etc.) with the hope of discovering a small number of molecules that are exquisitely diagnostic for the condition of interest. One discovery strategy that has been frequently used is to fractionate the biological samples being studied into simpler aliquots that can be more easily characterized using existing technologies. One such fractionation method is to isolate a specific portion based on a specific property (i.e., size, phosphorylation state, charge, etc.) of the proteins within the sample. This method provides a simplified sample that can be characterized at a higher coverage level than the complex sample from which it was derived. This chapter details one of these methods, the extraction and analysis of the low molecular weight proteome of human serum.

An integrated serum proteomic approach capable of monitoring the low molecular weight proteome with sequencing of intermediate to large peptides

The low-abundance, low molecular weight serum proteome has high potential for the discovery of new biomarkers using mass spectrometry (MS). Because the serum proteome is large and complex, defining relative quantitative differences for a molecular species between comparison groups requires an approach with robust separation capability, high sensitivity, as well as high mass resolution. Capillary liquid chromatography (cLC)/MS provides both the necessary separation technique and the sensitivity to observe many low-abundance peptides. Subsequent identification of potential serum peptide biomarkers observed in the cLC/MS step can in principle be accomplished by in series cLC/MS/MS without further sample preparation or additional instrumentation. In this report a novel cLC/MS/MS method for peptide sequencing is described that surpasses previously reported size limits for amino acid sequencing accomplished by collisional fragmentation using a tandem time-of-flight MS instrument. As a demonstration of the approach, two low-abundance peptides with masses of approximately 4000-5000 Da were selected for MS/MS sequencing. The multi-channel analyzer (MCA) was used in a novel way that allowed for summation of 120 fragmentation spectra for each of several customized collision energies, providing more thorough fragmentation coverage of each peptide with improved signal to noise. The peak list from this composite analysis was submitted to Mascot for identification. The two index peptides, 4279 Da and 5061 Da, were successfully identified. The peptides were a 39 amino acid immunoglobulin G heavy chain variable region fragment and a 47 amino acid fibrin alpha isoform C-terminal fragment. The method described here provides the ability both to survey thousands of serum molecules and to couple that with markedly enhanced cLC/MS/MS peptide sequencing capabilities, providing a promising technique for serum biomarker discovery.

Serum Proteomic Abnormality Predating Screen Detection of Ovarian Cancer

Ovarian cancer is characterized by vague, non-specific symptoms, advanced stage at diagnosis and poor overall survival. A nested case control study was undertaken on stored serial serum samples from women who developed ovarian cancer and healthy controls (matched for serum processing and storage conditions as well as attributes such as age) in a pilot randomized controlled trial of ovarian cancer screening. The unique feature of this study is that the women were screened for up to 7 years. The serum samples underwent prefractionation using a reversed-phase batch extraction protocol prior to MALDI-TOF MS data acquisition. Our exploratory analysis shows that combining a single MS peak with CA125 allows statistically significant discrimination at the 5% level between cases and controls up to 12 months in advance of the original diagnosis of ovarian cancer. Such combinations work much better than a single peak or CA125 alone. This paper demonstrates that mass spectra from the low molecular weight serum proteome carry information useful for early detection of ovarian cancer. The next step is to identify the specific biomarkers that make early detection possible.

Cancer biomarker discovery via low molecular weight serum proteome profiling – Where is the tumor?

Time-course analyses of rapidly processed serum performed in parallel by SELDI and nanoscale LC-MS/MS have revealed the temporal correlation of several literature-based disease markers with ex vivo driven events such that their in vivo existence in healthy subjects is questionable. Identification by MS/MS reveals these putative biomarkers to be byproducts of the coagulation cascade and platelet activation and suggests plasmatic analysis may be preferred. In a pilot plasmatic study, a cohort of naïve prostate cancer (PCa) samples were uniformly distinguished from their age-matched controls (n = 20) on the basis of multiple peptidic components; most notably by a derivative of complement C(4) at 1863 m/z (GLEEELQFSLGSKINVK, C4(1353-1369) ). The fully tryptic nature of this and other putative PCa discriminants is consistent with the cleavage specificity of common blood proteases and questions the need for tumor-derived proteolytic activities as has been proposed. In light of the known correlation of disregulated hemostasis with malignant disease, we suggest the underlying differentiating phenomena in these types of analyses may lie in the temporal disparity of sample activation such that the case (patient) samples are preactivated while the control samples are not.

Quantitative Analysis of the Low Molecular Weight Serum Proteome Using 18O Stable Isotope Labeling in a Lung Tumor Xenograft Mouse Model

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.